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Cell : The Unit of Life, , , , , , , , Microscopy, , Microscopy (Gk. Micros = small ; skopein = to see) is practice, of using microscopes for the study of finer details of small objects, including cells and tissues. Microscope are instruments consisting of, lenses (made of glass / Lithium fluoride / electromagnetic lens) which, ‘magnify and resolve small objects not visible to unaided eye for, the study of their details. The term microscope was coined by, Faber in 1625., , , , , , Magnification : It is the degree of enlargement or the ratio, of size of the object as seen in the microscope to its actual size, , Size of the image with the instrument, , Magnification = “5 of the Image with unalded eye, , Magnification of a microscope is roughly equal to the multiple, of magnifying power of objective lens and ocular lens (eye piece), e.g,, if the magnification power of an ocular lens is 10X and of the, objective is 40%, then the total magnilying power of a microscope, is 10x40=400X (the magnification power of a microscope is, represented by the symbol 'X')., , Resolving power : It is the ability of a system to distinguish, two close objects as two distinct objects, Its value is calculated by, Abbe equation —, , 061i, NA, , , , Here, A—is wavelength of used light, NA—Numerical, , Aperture, (INA =nsind), , , , Numerical aperture is multiple of refractive index of medium, (n) and sind, which is sine of angle substended by optical axis, and outer ray covered by objective. The value for best objective, 94,, , , , sine70, , Resolving power of a light microscope ranges from 0.2m to, 0.4m in blue light., , ‘The resolving power of human eye is 100m or microns (0.1, mm). This means that two points less than 100mm apart appear as, one point to our eves,, , Father of microscopy is Leeuwenhoek, He built first 270 X, magnification microscope in 1672, , Types of microscopes, , (1) Simple microscope : It is also known as magnifying, glass and consists of a convergent lens., , Leeuwenhoek (1683) designed a primitive microscope and, discovered cells with it. It was the first tool ever used to observe, biological objects. Its magnification power was 14 ~ 42 times only,, so itis considered as simple microscope., , (2) Compound microscope or Light microscope : The, first compound microscope was assembled by Zacharias Janssen, and J. Janssen, the Dutch spectacles makes in 1590. The, compound microscope was prepared by Kepler and Galileo in, 1611. However, it was not used for laboratory study. It is, simplest, widely used microscope having three lens ie,, condensor, which collects the light rays and precisely focuses, them on the objects; objective lens, which magnifies the image by, three objective lenses, ie. low power (10x), high power (45x), and oil immersion lenses., , Ina compound micrascope an object can be magnified upto, 1000 times and the magnification is independent of intensity of, light, size of microscope and numerical aperture. The light, microscope is also called bright field microscope because it forms, the image when light is transmitted through the object.

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486 Cell : The Unit of Life, , , , , , (3) Fluorescent microscope : It was developed by Coons, (1945). It is observed that when ultraviolet light is irradiated on, certain chemical substances, they absorb it and emit visible light, These chemical substances are called fluoro-chromes. The, fluorescent substances e.g, quinine sulphate, rhodamine and, auramine are used to stain the cellular objects and these objects, are easily visible as fluorescent areas when illuminated with, ultraviolet light., , , , (4) Polarizing microscope : It was invented by Tolbart. In, this microscope the plane polarised light is used as a source of, illumination. Unlike the ordinary light, plane polarised light vibrates, only in one direction and the cellular objects are easily visible as, they appear bright against the dark ground. Polarizing microscope, is helpful in studying the spindle fibres in the cells., , , , (5) Ultraviolet microscope : It was invented by, Cospersson. In this microscope the source of illumination is, ultraviolet radiations having shorter wavelengths (1500 A —, 3500 A) as compared to ordinary visible light. In this, microscope, the lenses are made of fluoride, lithium fluoride or, quartz instead of glass, , , , , , Ultraviolet microscope is helpful in quantitative determination, of all those cell components which absorb ultraviolet rays., , (6) Phase contrast microscope, {i) Discovered by Dutch man Fredericke Zernicke (1935),, (ii) Source of, , , , rmination is visible light., , (ii) It is used to study living cells and tissues without staining, and effect of chemical and physical agents on the living cells,, , {iv) It is also used to study spindle formation, pinocytosis,, karyokinesis, cytokinesis etc., , (v) The demerit of this microscope is subcellular organelles, smaller than 0.211, (like ribosomes, lysosomes, ER) cannot be, , visualised., (7) Interference microscope (Morten et.al.), , (i) Ws principle is similar to that of the phase contrast, microscope and gives / studies quantitative data., , (i) Nomarski interference contrast microscope is useful to, study mitosis /cell components in living state,, , (ii) It gives better image of living structures. It is also used to, measure thickness of the cell and determination of several light, absorbing chemicals like nucleic acid, proteins, lipids etc., , (8) Dark field microscope, {i) Zsigmondy (1905) invented this microscope., , (ii) It is based on the fact that light is scattered at boundaries, between regions having different refractive index., , (iii) The object smaller than those seen with ordinary light, microscope can be detected but can not be resolved., , , , (9) Electron microscope : This was developed by M. Knoll, and E. Ruska (1931) in Germany. Itis the best device to determine, the ultrastructure of a cell organelle, It is a large sized instrument, which has an intemal vacuum, high voltage (50,000 - 1,00,000, volts), a cooling system, a fast beam of electrons (0.54 A, wavelength}, a cathod filaments of tungsten and electromagnetic, lens (which having a coil of wire enclosed in soft iron casing) for, focusing Ribosomes can be seen only in electron microscope., , ‘Thus an electron microscope essentially comprises an electron, gun and electron lenses. The electron gun is the source of electrons, consisting of a heated tungsten filament. It is preferred because it, can be heated upto 300°C. The electron beam can be reflected by, magnetic field. Therefore, a very powerful magnetic coil acts as, lens. The focal length of the electromagnetic lenses change with the, wavelength of illumination, Since the wavelength is controlled by, the voltage, it should be controlled and made constant. Three, types of magnetic lenses are used namely projector, objective and, condenser. The magnetic field produced is concentrated by soft, iron casing, When the filament is heated to incandescence, it emits, electron. The electrons then move to positively charged anode., The entire microscope column operates under conditions of high, vacuum. It is due to this fact that we can not observe living objects, through an electron microscope (EM). For viewing objects under, EM, ultrathin sections (20-100 nm thick) are prepared through an, ultramicrotone, It was first developed by W.His., , Electron microscope can magnify the objects upto 2,00,000, 1es (now possible upto 2,50,000 - 4,00,000) and direct study of, objects is possible on this microscope. The resolving power of, electron microscope is 10 A which is 100 times more than the light, microscope. Study of living cells can not be done through this, microscope because of high voltage, which is required to operate, it, ill the living materials. Electron microscope are of two types :, , (i) Transmission electron microscope (TEM) : It was the, first microscope developed by Ruska (1932). It produces two, dimensional images., , Magnification of TEM is 1-3 lakh and resolving power is 210A, Because of them transmission electron microscope has, helped in the discovery of a number of small cell organelles e.g.,, ER, ribosomes, centrioles, microtubules etc. Detail structure of, larger cell organelles could also be known only with the help of, TEM, eg, chloroplast (thylakoids), mitochondria (elementary, patticles, DNA), ribosomes etc., , (i) Scanning electron microscope, microscope was invented by Knoll (1935)., , (SEM) : This, , It gives three dimensional image. The specimen to be studied, is first super cooled (in liquid propane at -180°C) and dehydrated, in alcohol (at 70°C), It is then coated with gold, platinum or some, other metals for creating a reflecting surface for electrons,, ‘Magnification of SEM varies from 15 ~ 2,00,000. Resolution power, is 5-20 nm.

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(10) Advanced high power micr, , , , ‘ope, , () Scanning probe microscope : The microscope is, capable of resolving the outer texture of the material to the, minutest detail since it has the potential to image even a single, atom. Magnification is upto 100 million, , (ii) Scanning tunnelling microscope : It has a tiny, tungsten probe for moving over the surface of specimen. The, microscope is used to detect defect in clecrical conductors and, computer chips, , (il) Atomic force microscope : It has an extremely fine, diamond probe for moving over the surface of biochemicals, Oscillations produced in the probe are changed into images by a, computer., , The microscope is useful in viewing detalled structure of, biological molecules, e.g., DINA, proteins, etc., , Units of measurement used in microscopy, 1 micron (u) = 10°* or one millionth, 1 micrometer (4am) = 10% m, 10% em, 10° mm =1000nm, 1 Nanameter (nm)=10-%m, 107 em 10° mm,10° 2m =10A, 1 Angstrom (A)= 10°" m, 10 cm, 10° mm, 107 ym,, 1 Picometer (pm) = 10" m,10-% nm, 1 Femtometer (fm) = 10-°m,10nm, 1 Attometer = 107m,10°nm, , ‘Common unit of measurement in Microscopy and cytology is, nanometer while unit of measurement of cell is micron., Cytochemistry, , ‘A number of dyes or stains are known to colour specific parts., Certain dyes can be used even in case of living materials. They are, called vital stains, ¢.g,, neutral red, methylene blue., , , , Fuelgen or Schiff's reaction was developed by Fuelgen and, Rossenbeck (1924). Identification and localization of chemical, compounds of a cell is studied in cytochemistry,, , , , , , , , , , , , , , , , , , , , , , , , Table : 3.1-1 Some important cytochemical stains, Stain Used for staining Final colour, ‘Aceiocarmine Chromosomes Pink, ‘Acid fachsine Coriex, cellular walls, Magenta, mitochondria, Aniline blue Fungal hyphae Blue, Basic fuchsine Nucleus ‘Magenta red, Crystal violet Bacteria Violet, Eosin Cytoplasm Pink, Feulgen’s sain DNA) Purple/Red, Hemotoxyline Nuclei cel wall cellase | Violet, Iodine solution Starch Blue, , , , , , , , Cell : The Unit of Life 487, , , , , , , , , , , , , , , , , , , , , , Janus green Fungi and mitochondria | Green, Methylene blue ‘Yeast and Golgi complex. | Blue, Phloroaiucinol +FICI | Lignin Red, Rutheniumzed | Pectin Red, Safranin Nuclei ignified sue | Red, ‘Sudan- lil or 1V ‘Suberin, cutin, olf Red, ‘Sudan black Fatty substance Black, Toludine blue RNA Blue, Cotton blue Fungi Violet, , , , , , , , Cell fractionation, , In isotonic medium cell components are separated, in two, steps process., , Homogenisation : Cell products are separated in isotonic, medium (0.25 M sucrose solution) either with the help of, homogeniser of ultrasonic vibrations kept at 0 - 4°C. A homogenised, cell is called homogenate, , Differential centrifugation : Homogenisation product is, rotated (centrifuged) at different speeds. The sediment or pellete of, each speed is collected. eg., nuclei at 1000xg (g= force of, gravity) for 10 minutes, chloroplast and mitochondria at, 10,000xg for 15 minutes, The particle settle according to their, sedimentation ratios. Sedimentation coefficient is expressed in, svedberg unit ‘S' related with molecular weight of the particles. For, the detail study of mitochondria it is the best technique. 'S' is, measured by analytical centrifugation., , The various cell organelles and macromolecules sediment in, the following order., , ‘Nucleus~sChloroplast—+Mitochondria—»Ribasome~»DNA-—>mRNA—tRNA, Chromatography, , Discovered by Michael Tswett (1906). This technique is used, to separate the molecules of different substances present together., Mixture of molecules is run over an adsorption medium, Chromatography may be following types, , , , Adsorption or Column chromatography : The stationary, phase consists of a column of charcoal, silica, alumina, calcium, carbonate or magnesium oxide. The solution is made to, percolate through this column when different chemicals get, absorbed at various levels. The technique is useful for separation, of tissue lipids,, , Thin layer chromatography (TLC): The stationary phase, consists of a thin plate of cellulose powder or alumina. As a few, drops of mixture are poured over it, the different chemicals spread, to different distances. The method is useful in separation of amino, acids, nucleotides and other low molecular weight products, , Paper chromatography : A paste of mixture is applied near, one end of a chromatographic paper (or Whatman 1). The lower, end below the paste is dipped in a solvent. As the solvent rises in, chromatographic paper, the different chemicals of the mixture, spread to different distances. The paper can be rotated to obtain, two dimensional chromatogram.

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488 Cell : The Unit of Life, , , , ‘Types : (a) Ascending (b) Descending (c) 2-D chromatography., , Ton exchange chromatography : Beads of cellulose and, other materials having negative and positive charges are placed in, a column. The mixture (mobile phase) is poured over the column., |As the mixture passes through the column, its constituents separate, according to their charges. The technique is used in purification of, insulin, plasma fractionation and separation of proteins., , Gel fractionation / Gel filtration chromatography, (Molecular sieve chromatography) : The stationary phase, consists of gel forming hydrophilic beads which contain pores,, eg, sephadex (cross-linked dextran). As the mixture is poured, over the gel, larger molecules pass out unimpeded while small, molecules are trapped in the pores. The technique is used in, separation of proteins. It is also employed in determining their, molecular weight by calibrating the column with proteins of, known molecular weight., , , , Affinity chromatography : Stationary phase consists of, column of ligands (molecules that bind to other specific molecules, at particular sites)., , Mixture is allowed to pass through the column. Chemical, linkages are established between ligands and their specific, chemicals. Others pass out of the column. The technique is used in, separation of enzymes, immunoglobulins, mRNA, etc, , Electrophoresis, , It is an another technique of separation in which patricles of, different charges and sizes are separated under the influence of, electric field. e.g., nucleic acids, proteins, amino acids, nucleotides, can be separated by this method. The technique was discovered by, Russian physicist Alexender Reuss in 1807., , Autoradiography, , Ttis a technique of studying the route of chemicals in chemical, reactions taking place inside the cell and organisms with the help of, radioactive isotope. e.g., "C,H, *P., , In this technique the radioisotopes are incorporated into the, precursor molecule. Then the labelled precursor molecules, roduced into the cells and their path is followed with the help of, thelr radiations., , , , Radioactive precursors emit radiations and their positio:, the cell is located by bringing the cell in contact with a, photographic plate or film., , , , ™P and “'C are used for the study of nucleic acids and, photosynthesis (Melvin Calvin) respectively, , Radioisotope or Tracer tech, , ‘They are unstable isotopes which function like normal elements, ‘but emit positive or negative particles, e.g., 7H (Tritium), “C (Carbon),, =P (Phosphorus), *S (Sulphur), #K (Potassium), I (Iodine), Radioactivity is recorded in different parts by Geiger counter or, scintilation counter or autoradiography to know regions of use and, transport. The tracers have been used for knowing pathway of mineral, transport (Stout and Hoagland, 1939), organic solute transport, (Vemon and Aronoff, 1952), carbon assimilation (Calvin, 1955)., , jue, , , , Where radioactive elements are not available, heavy isotopes, are used, eg, “N, "O, Their fate is recorded by mass, spectroscopy and density gradient centrifugation. Meselson and, Stahl (1958) studied DNA replication and Ruben et al (1941), evolution of oxygen (photolysis of water) in photosynthesis by, using heavy isotopes., , %ray crystallography, , It was developed by the Bragg (1913). They can be used as a, tool for determining the arrangement of atoms in various biological, molecules,, , By using this technique Wilkins et al, 1953 found out details, of the DNA molecule for which he was also awarded Nobel Prize, along with Watson and Crick in 1962, Kendrew, 1957 by using the, same technique studied the molecules of myoglobin., , Cell as a unit of life, , Cytology (Gk Kytos = cell ; logos = study) : It is the branch of, biology, which comprises the study of cell structure and function., “Cell is the structural and functional unit of all living beings”. Study, of metabolic aspects of cell components is called cell biology., , Robert Hooke (1665) discovered hollow cavities (empty boxes), like compartments in a very thin slice of cork (cell wall) under his, microscope. He wrote a bock “Micrographia” and coined the term, cellula, which was later changed into cell. Grew and Malpight also, observed small structures in slice of plants and animals, Leeuwenhoek, was the first to see free cells and called them “wild animalcules” and, published a book “The secret of nature’. He observed bacteria,, protozoa, RBCs, sperms, etc. under his microscope., , Cell theory : H.J. Dutrochet (1824) a French worker gave, the idea of cell theory,, , , , ‘The actual credit for cell theory goes to two German scientists,, a Botanist Mu. Schleiden (1838) and a Zoologist T. Schwann, (1839). They gave the concept “all living organisms are composed, of cell”. Schleiden and Schwann both supported the theory of, “spontaneous generation”. They also mentioned that “the new cell, arises from nucleus by budding”., , Exceptions to the cell theory : Viruses, viroids and prions, ‘are an exception to the cell theory as they are obligate parasites, (sub-cellular in nature), , Modification of cell theory : Modification of cell theory, was done by Rudolf Virchow (1855). He proposed the “law of cell, lineage” which states that cell originates from pre-existing cells. 1e.,, (omnis cellula-e-cellula). It is also called “cell principle” or “cell, doctrine”. It states, , (1) Life exists only in cells, , (2) Membrane bound cell organelles of the protoplasm do not, survive alone or outside the protoplasm., , (3) Cells never arise de novo. The new cells are like the parent, cell in all respect., , (4) All cells have similar fundamental structure and metabolic, reactions., , (5) Cells display homeostasis and remain alive.

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(6) Genetic information is stored in DNA and expressed within, the cells,, , (7) DNA controls structure and working of a cell., , The cell as a self contained unit : Autonomy of a cell is, believed due to presence of DNA and its expressibility, otherwise, cell, components have different shape and function. It has two positions,, , (1) Autonomy in unicellular organisms : Unicellular, ‘organisms leads to a totally independent life due to different shape,, size and role of different organelles shows division of labour. All, these display homeostasis. Unicellular organisms are more active, due to large surface volume ratio,, , (2) Autonomy in multicellular organisms : In, ‘multicellular organisms life activities are displayed by each of the, cells independently, Multicellular organisms have one thing, advantage over unicellular organisms is division of labour., , Cellular totipotency : Totipotency was suggested by, Haberiandt (1902). When cells have tendency or ability to divide, , , , phenomenon of cellular totipotency in phloem tissue of carrot., , Surface volume ratio : Metabolically active ces are small, as, small cells have higher nucleocytoplasmic ratio for better control and, higher surface volume ratio for quicker exchange of materials, between the cell and its outside environment. Larger cells have, lower surface volume ratio as well as lower nucleocytoplasmic ratio., Surface volume ratio decreases if cel size increases., , Table : 3.1-2 Differences between plant and animal cell, , , , , , , , , , , , , , Plant cell ‘Animal cell, Coll wall present. Cell wall absent., Nucleus usually lies near periphery | Nucleus present near the centre,, due to vacuole, , , , Centrosome is usually absent from | Usually centrosome Is present, higher plant cells, except lower | that helps: in formation of, , , , , , , , , , , , , , , , , , , , , , , , , , motile cells spindle fibres., , Presid are present, exept fang. Plastids are absent, Mitochondria is generally spherical | Generally tubular in shape,, , or oval in shape,, , Single large central vacuole is | Many vacuoles occurs, which, resent. are smaller in size,, , Cytoplasm during cell division | Cytoplasm divides by furrowing, Usually divides by cell plate method. | or cleavage method., , Plant cells are capable of forming all | Animal cells cannot form all the, the amino acids, coenzymes and | amino acids, coenzymes and, vitamins, vitamins,, , There is no contractile vacuole. Contractile vacuole may occur, , fo pump excess water, , , , , , , , Spindle formed during cell division | Spindle formed during cell, , is anasral division are amphiastal, , Lysosomes present in ess number. | Lysosomes present in more, number,, , , , , , , , Cell : The Unit of Life 489, , , , Types of cells, , , , Chatton gave the term prokaryote and eukaryote. Depending, upon the nature of nucleus, cells are classified. Incipient nucleus is, Present in prokaryotes, where as in eukaryotes well organised, nucleus is present., , , , , , , , , , , , , , , , Table : 3.1-3,, Differences between prokaryotic and eukaryotic cell, Prokaryotic cell Eukaryotic cell, Itisa single membrane system. Its a double membrane systern., Cell wall surrounds the plasma | Cell wall surounds the plasma, ‘membrane, membrane In some protists,, most fungl and all plant cell,, Animal cell lacks it, Coll “wall is composed of [tis composed of, peptidodiyeans, Strengthening | polysaccharide, Strengthening, material is murein. ‘material is chitin in fungl and, , cellulose in other plants, It lacks respiratory enzymes., , , , Cell membrane: bears. respiratory, enzymes, (Cyioplasm lacks cell organelles e.g,, , , , Cytoplasm coniains various eel, , Mitochondria, ER. Golgi body ete. | organelles., , Ribosomes are only 705 type. Ribosomes are both 80 § and, 705 type., , There are no streaming movements | Cytoplasm show streaming, , of cytoplasm, movements,, , It is enveloped by nudear, envelope, Nucleus is distinct, from cytoplasm,, , Nuclear DNA is linear and, associated with histone proteins, extranuclear DNA is circular, and histone protein free., , Sextal reproduction is present., , Nadear matatal i not endosod by, rmudear ervelope and Kes dicey in, staples. tis elle nurleck, , DNA is circular and not associaled, with histone proteins,, , , , Sexual reproduction absent but, parasexuality present,, , Cell division mostly amitotic,, , , , , , Cell division is typically mitotic, Mesokaryon : Dodge gave the term ‘Mesokaryon’ for, , dinoflagellates. These are intermediate type of cell organisation in, , dinophyceae of algae. In mesokaryotic there is present a true or, , eukaryotic nucleus with definite nuclear membrane and, , chromosomes., , Cell wall, , Discovery : It was first discovered by Robert Hooke in 1665, in Cork. Cell wall is the outer most, rigid, protective, non living and, supportive layer found in all the plant cells, bacteria, cyanobacteria, and some protists. It is not found in animal cells, , , , ‘Chemical composition : Mainly cell wall consists of two, parts, matrix and cellulosic fibres (microfibrils). Matrix consists of, hemicellulose, pectin, glycoproteins, lipids and water. In most of, the plants cell wall is made up of cellulose (C,HcO;),,a polymer, made-up of unbranched chain of glucose molecule linked by, BA1=4 glycosidic bond. About 100 molecules of cellulose form a, micelle, about 20 micelle form a microfibril and approx. 250, microfibril form a fibril