పిల్లులు తాము వయస్కులుగా నిలబడాలంటే 7 ఇండ్లు మరలట. మన ఈ టివి పరకాలను చుస్తే కూడా ఇదే గుర్తుకు వస్తుంది. ఏ వక్కరినీ మాట్లాడనీయక తనే మాట్లాడుతూ, తను అనుకున్న విషయాన్నే అందరూ మాట్లడలే తప్ప వేరే మాటలు మాట్లాడ వద్దన్నట్లుగా ఉంటుంది ఈయనగారి చర్చావేదిక. ఇక ఇప్పుడేమో విశాలాంద్ర కావాలట. మరి సారుగారు అనుకున్నదే అందరు చేయాలిగా, అందుకే విక్రమార్కునిలా మొదలెట్టాడు యాత్రను. మరి ఏం సాదించారు అనుకుంటున్నారా. లేదులెండి ఆయన సాదించారు 'తన్నిన్చుకొన్నారుగా మరి'. హ హ హ హ....
Monday, November 14, 2011
Sunday, August 15, 2010
MICRO SCOPE
Microscopy
Microscope is the basic, routine and most characteristic tool or instrument for scientists in laboratories. The main function of any microscope is the magnification of the object. The magnification that is attainable by microscopes ranges from 100 to X 400,000. In addition to the magnification, a microscope must possess good resolution or resolving power which is the ability to distinguish two adjacent points as distinct and separate.
Different types of microscopes are available today and each of them are having their own advantages and disadvantages. The microscopes used today have evolved significantly from Leevwenhoek's first simple microscope.
Depending upon the magnification principle involved, microscopes are of two main categories. They are
1. Light microscope
2. Electron microscope.
4.1.1. Light Microscope
Light microscopes use optical lenses and visible light for magnification. The light microscope can magnify images up to about 1000 times. To produce an ideal magnified image, the light microscope must possess a good resoving power and suitable numerical aperture. There are two types of light microscopes namely i) Simple microscopes and ii) Compound microscopes.
i) Simple microscope :
A simple microscopes consists only one biconvex lens.
ii) Compound microscope :
Compound microscopes are more complex but are common in usage today. A compound microscope consists of atleast two lens systems viz., objective and ocular or eye piece. The objective lens lies close to the specimen or object and magnifies the specimen. The ocular further magnifies the image produced by the objective lens. So, the total magnification obtained in a compound microscope is equal to the product of the magnifying power of the two sets of lenses. The magnification of the specimen through this compound microscope ranges from 1000 to 2000 times the diameter of the specimen.
A common compound microscope used today consists a series of optical lenses, mechanical adjustment parts and supportive structures for these components. The optical lenses include ocular, objectives and the substage condenser. The number of objectives is usually three with different magnifying powers. The function of condenser is to collinate the light rays in the plane of the microscopic field. The mechanical adjustment parts comprise the coarse, fine, and condenser adjustment knobs. The supportive structures include the base, arm, pillar, body tube or barrer and resolving nosepiece.
The most commonly used compound light microscopes are
1. Bright field microscope. 2. Dark field microscope
3. Phase contrast microscope 4. Fluorescent microscope
1. Bright field microscope : The bright field microscopes are the most commoly used microscopes in biology and microbiology courses. If forms a dark image of the specimen against a bright background. As a result, in bright field microscopy, the microscopic field is brightly lighted and the microorganisms appear darker because they absorb some of the light. Generally, most of the microorganisms do not absorbing ability increases. As a result, they absorb more light and exhibit greater contrast and colour differenciation. The basic limitation of the bright - field microscope is the resolving power. Normally, they produce a useful magnification of about 1000 to 2000. At a magnification greater than 2000 the image becomes fuzzy in their microscopy due to less resolution. At the best, the resolving power of this bright - field microscope is 0.2mm. That means, a bright field microscope can clearly distinguish two objects separated by distance of 0.2 mm.
2. Dark field microscope : In this microscope, the specimens or objects are brilliantly illuminated against the dark background of microscopic field. This dark - field microscope consists a special kind of condenser called as 'Abbecondenser' which is having an opaque disk or dark field stop. This special condenser transmits a hollow cone of light from the source of illumination. As a result, most of the light directed through the condenser does not enter the objective and thereby the microscopic field becomes dark. In dark - field microscopy, only the light rays deflected or scattered by the specimen cuts on light same for eye piece so the specimen appear as a bright object on a dark backgroud. Dark - field microscopy is particularly valuable for the examination of unstained microorganisms suspended in fluids such as wet mount and hanging - drop preparations.
3. Phase-contrast microscope : This is special microscope which permits the study of unstained objects The phase- contrast microscope consists of a phase - contrast objective and a phase. The phase contrast microscope is an ordinary bright field microscope with two additional plates, namely annular diaphram and phase shifting plate. The phase contrast principle was discovered by Fritz zernike who was awarded Noble prize in physics in 1953. According to this principle, light waves have variable character for frequency and amplitude. Human eyes can not perceive a phenomenon when two light rays have similar amplitude and frequency but different phase. This special optical system makes it possible to distinguish unstained structures with in cell that differ slightly in their thickness. The phase of light is altered by different components of a cell e.g. the nucleus and cytoplasmic granules depeding on alterations of phase are converted into optical densities. Thus an unstained object like an animal cell, when viewed through this system, shows a wealth of structural detail not visible through the ordinary microscope.
Phase - contrast microscopy is extremely useful and valuable for studying living unstained cells is widely used in applied and theoretical biological studies.
4. Fluorescence micro scope : The main principle involved in this fluorescent microscopy is the phenomenon of fluoresence. Some chemical substances absorb light of a particular wave length and energy and then emit light of longer wave lenght and lesser energy. Such substances are called as fluorescent substances. The phenomenon is known as fluoresence. The fluorescent microscope is used to visualize specimens that naturally fluoresence as they contain fluorescent substances. For example, chloropyll fluoresces brilliant red. Specimens can also be observed in this microscope by treating the specimens with fluorescent dyes like acridine orange R, auramine O, primulin, thiazo - yellow G etc. These fluorescent dye molecules are called as fluorochromes.
In practice, the microorganisms are stained with a fluorescent dye and the illuminated with blue light. The dye absorbs the blue light and emit green light.The fluorescence microscope consists of special components like exciter filter and barrier filter for the purpose. The function of the exciter filter is to remove all but the blue light. The barrier filter functions in blocking the blue light and allowing green light to pass through and reach eye. The selection of barrier filters depends on the dye used in microscopy.
The best adoption of this microscopy is Immuno-fluorescence’. Antibodies can be chemically combined with fluorescent dyes such as fluorescein isothiocyanate and lissamine rhodamine B. The antibodies. these labelled antibodies when mixed with a suspension of bacteria gets attached to the surface of the bacteria. These bacteria with attached labelled antibodies are clearly visible when observed by fluorescent microscopy. This procedure is known as fluorescent antibody technique and thephenomenon is called as immunofluorescence.
The fluorescence microscope is an essential and useful tool in medical microbiology and microbial ecology. Bacterial pathogens like Mycobacterium tuberculosis are easily identified by suing fluorochromes or fluorescent antibody technique.
Inverted Microscopes
There are two basic types of microscopes. The one most people are familiar with looks down at the specimen with the light source coming from below and is called an upright microscope. An inverted microscope looks up at the specimen with the light source coming from above instead.
Inverted microscopes were first invented in 1850 by Tulane University's J. Lawrence Smith and debuted at the World's Fair in London in 1852. In the early 20th century, they began to be used for observation of living cells, particularly for aquatic life. It was also used for analysis of heavy metals like iron and steel before World War II.
As the name suggests, an inverted microscope is upside down compared to a conventional microscope. The light source and condenser are on the top above the stage pointing down. The objectives and turret are below the stage pointing up.
The only things that are "standard" are that
1) a specimen (as dictated by the laws of gravity) is placed on top of the stage and
2) thank heavens, the binocular or trinocular tube is not upside down but in the standard position pointing at a conventional viewing angle. As a result, one is looking up through the bottom of whatever is holding the specimen and is sitting on the stage rather than looking at the specimen from the top, typically through a cover glass, as on a conventional microscope.
Grades of inverted microscope :
There are two grades of inverted microscopes.
1. A routine inverted microscope is small and comes in low and medium power settings. These can be used in homes and small labs in schools. They are limited in what the can observe as they usually do not allow for fine focus and have relatively low power magnification.
2. A research inverted microscope comes in heavy power settings and can allow for a very fine focus. The major disadvantage to them is that they are extremely expensive and are usually only used by universities and medical institutions. They are usually able to accommodate video cameras and televisions to assist in research documentation. The improvements on the inverted microscope over the course of the 20th and 21st centuries have allowed it to be an integral part of advanced scientific research.
Advantages :
1. An inverted microscope is most helpful when looking at heavy objects or those which are greatly effected by gravity. Material specimens like metal can be large and heavy. They require the large staging areas that inverted microscopes allow for.
2. The materials greatly affected by gravity include living cells and aquatic life that tend to pool and collect on the bottom of specimen containers. An inverted microscope looks at the sample from the bottom, making it easier to see the organisms with ease. It also allows users to see the samples in a more natural environment than a standard glass slide. Petri dishes allow more movement for the samples and are commonly used with inverted microscopes.
3. This type of microscope has been redesigned and improved on to accommodate particular uses. There are stages made particularly for processes like incubation and in vitro fertilization. The nosepieces have been made larger and revolvable, making to make it easier for scientists to identify and rotate objects. They have also been made heavier and sturdier, allowing for less vibration and greater ease of observation.
4. Inverted microscopes are often used for looking at living organisms and tissue that may be killed by staining, they often provide for "optical staining" through the use of phase contrast or DIC. My CK2 has phase contrast. Rather than use the more sophisticated phase contrast condenser used on a standard microscope, there is a simple slider that goes through the condenser and holds the necessary phase rings. Only the phase rings for the lower power objectives (e.g. 10x and 20x) are centerable by a fairly crude process (although it works). The phase ring for the 40x objective is not but seems to work fine.
4.1.2 Electron Microscope
Electron microscopes are of more recent origin and more sophisticated and useful than high microscopes. For the first time, Knoll and Rusca developed the electron microscope in 1932. The electron microscopy markedly differs in many respects from the optical microscopic technique. Electron microscope uses a beam of electrons and magnetic field as light and optical lenses, respectively, and the whole system operates in high vacuum. The electron microscope provide tremendous useful magnifications, because of much high resolution obtained than in light microscope.
As the electron beam has extremely short wavelength of only 0.05 A it provides much greater resolving power. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to x 40,000. There are two types of electron microscopes namely.
1. Transmission electron microscope ( TEM)
2. Scanning electron microscope (SEM)
1. Transmission electron microscope ( TEM) : The modern Transmission electron microscope is a complex and sophisticated microscope. The source of illumination in TEM is the electron gun which is made up of by a thin, V- shaped tungsten filament. On heating, the tungsten filament generates a beam electrons which is then focused on the specimen by the condenser. The specimen scatters the electrons passing through it. Then the beam is focused by objective magnet lens and project magnet lens to form an enlarged, visible image of the specimen on fluorescent screen. The magnified image can also be recorded on a photographic plate by a camera built into the instrument. Some substances are denser absorbing more electrons than others.
Usually, the penetrating power of the electrons through solid matter is weak. So, the specimens are prepared as either thin films or thin sections. To increase the contrast of these thin preparations of the biological specimens, they are usual treated with special electron microscopic stains. The important stains are osmic acid, permanganate, uranium, lanthanum and lead.
Specimen preparation : In order to observe specimens such as isolated macromolecules of protein or nucleic acid or to study the structural features of cells there are special techniques for specimen preparation. Two important techniques for specimen preparations are shadow casting and freeze etching.
i) Shadow casting technique : In this, the dried specimen is deposited with electron dense or heavy metal at an angle of about 45 from horizontal. The heavy metals normally used for this purpose are platinum, chromium, nickel, uranium and alloys of gold and platinum or platinum and palladium. This shadow casting technique increases the specimens contrast. This technique is particularly useful in studying virus morphology, bacterial flagella.
ii) Freeze etching technique : This is the more recent technique which avoids the fixation, embedding and sectioning of specimens. In this technique, the specimen is rapidly frozen in liquid nitrogen and then fractured with a knife pre-cooled with liquid nitrogen. The exposed surfaces of the fractured specimen are shadowed and coated with layers of platinum and carbon to form a replica of the surface. Later, these carbon replicas of the surfaces are used to study the details through TEM. This technique is found more useful in studying cell wall and membrane structures.
Draw backs of TEM :
1. Material to be observed should be dehydrated as TEM operates in vacuum.
2. The dehydration and treatment of specimen with electron dense staining substances may bring about changes in original structure.
3. The specimen preparation should not be thick, as electrons cannot penetrate through dense or thick material.
2. Scanning electron microscope (SEM) : The scanning electron microscope is a comparatively new type of electron microscope. It is quite different in principle and application from transmission electron microscope. The SEM gives a three dimensional quality to specimen images. It is useful in studying the surfaces of microorganisms in great detail. It possess a great depth of focus and can be used to observe even fairly large specimens.
- With this scanning electron microscope a wide range of magnifications from a low as X 15 up to about 1,00,000 is possible. In this a narrow beam of electrons rapidly move back and forth across the surface of metal coated specimen and scan the surface features. In contrast to the transmission electron microscope, the electrons emitted by the object’s surface produce an image in SEM.
The SEM consists a detector, photomultiplier, cathode ray tubes for, viewing and photography as special components. In scanning electron microscopy, the specimen preparation is comparatively simple and, the material to be observed is coated with a thin layer of heavy metal such as gold. In SEM, the beam of electrons released from the electronic gun strike the specimen and scans the surface. During this scanning, secondary electrons are released from the specimen. These secondary electrons are collected by a detector where an electronic signal is generated. These signals are amplified by photomultiplier and then sent to a cathode ray rube which produces an image like in a television system. This image can be viewed and photographed. The SEM is especially useful in studies, of bacterial cells, spores, fungi and morphological changes in tissues infected with microorganisms.
Draw backs of SEM :
1. The resolving power of SEM is comparatively less than that of transmission electron microscope.
2. It gives only the surface characters of the specimen but not of internal components.
Microscope is the basic, routine and most characteristic tool or instrument for scientists in laboratories. The main function of any microscope is the magnification of the object. The magnification that is attainable by microscopes ranges from 100 to X 400,000. In addition to the magnification, a microscope must possess good resolution or resolving power which is the ability to distinguish two adjacent points as distinct and separate.
Different types of microscopes are available today and each of them are having their own advantages and disadvantages. The microscopes used today have evolved significantly from Leevwenhoek's first simple microscope.
Depending upon the magnification principle involved, microscopes are of two main categories. They are
1. Light microscope
2. Electron microscope.
4.1.1. Light Microscope
Light microscopes use optical lenses and visible light for magnification. The light microscope can magnify images up to about 1000 times. To produce an ideal magnified image, the light microscope must possess a good resoving power and suitable numerical aperture. There are two types of light microscopes namely i) Simple microscopes and ii) Compound microscopes.
i) Simple microscope :
A simple microscopes consists only one biconvex lens.
ii) Compound microscope :
Compound microscopes are more complex but are common in usage today. A compound microscope consists of atleast two lens systems viz., objective and ocular or eye piece. The objective lens lies close to the specimen or object and magnifies the specimen. The ocular further magnifies the image produced by the objective lens. So, the total magnification obtained in a compound microscope is equal to the product of the magnifying power of the two sets of lenses. The magnification of the specimen through this compound microscope ranges from 1000 to 2000 times the diameter of the specimen.
A common compound microscope used today consists a series of optical lenses, mechanical adjustment parts and supportive structures for these components. The optical lenses include ocular, objectives and the substage condenser. The number of objectives is usually three with different magnifying powers. The function of condenser is to collinate the light rays in the plane of the microscopic field. The mechanical adjustment parts comprise the coarse, fine, and condenser adjustment knobs. The supportive structures include the base, arm, pillar, body tube or barrer and resolving nosepiece.
The most commonly used compound light microscopes are
1. Bright field microscope. 2. Dark field microscope
3. Phase contrast microscope 4. Fluorescent microscope
1. Bright field microscope : The bright field microscopes are the most commoly used microscopes in biology and microbiology courses. If forms a dark image of the specimen against a bright background. As a result, in bright field microscopy, the microscopic field is brightly lighted and the microorganisms appear darker because they absorb some of the light. Generally, most of the microorganisms do not absorbing ability increases. As a result, they absorb more light and exhibit greater contrast and colour differenciation. The basic limitation of the bright - field microscope is the resolving power. Normally, they produce a useful magnification of about 1000 to 2000. At a magnification greater than 2000 the image becomes fuzzy in their microscopy due to less resolution. At the best, the resolving power of this bright - field microscope is 0.2mm. That means, a bright field microscope can clearly distinguish two objects separated by distance of 0.2 mm.
2. Dark field microscope : In this microscope, the specimens or objects are brilliantly illuminated against the dark background of microscopic field. This dark - field microscope consists a special kind of condenser called as 'Abbecondenser' which is having an opaque disk or dark field stop. This special condenser transmits a hollow cone of light from the source of illumination. As a result, most of the light directed through the condenser does not enter the objective and thereby the microscopic field becomes dark. In dark - field microscopy, only the light rays deflected or scattered by the specimen cuts on light same for eye piece so the specimen appear as a bright object on a dark backgroud. Dark - field microscopy is particularly valuable for the examination of unstained microorganisms suspended in fluids such as wet mount and hanging - drop preparations.
3. Phase-contrast microscope : This is special microscope which permits the study of unstained objects The phase- contrast microscope consists of a phase - contrast objective and a phase. The phase contrast microscope is an ordinary bright field microscope with two additional plates, namely annular diaphram and phase shifting plate. The phase contrast principle was discovered by Fritz zernike who was awarded Noble prize in physics in 1953. According to this principle, light waves have variable character for frequency and amplitude. Human eyes can not perceive a phenomenon when two light rays have similar amplitude and frequency but different phase. This special optical system makes it possible to distinguish unstained structures with in cell that differ slightly in their thickness. The phase of light is altered by different components of a cell e.g. the nucleus and cytoplasmic granules depeding on alterations of phase are converted into optical densities. Thus an unstained object like an animal cell, when viewed through this system, shows a wealth of structural detail not visible through the ordinary microscope.
Phase - contrast microscopy is extremely useful and valuable for studying living unstained cells is widely used in applied and theoretical biological studies.
4. Fluorescence micro scope : The main principle involved in this fluorescent microscopy is the phenomenon of fluoresence. Some chemical substances absorb light of a particular wave length and energy and then emit light of longer wave lenght and lesser energy. Such substances are called as fluorescent substances. The phenomenon is known as fluoresence. The fluorescent microscope is used to visualize specimens that naturally fluoresence as they contain fluorescent substances. For example, chloropyll fluoresces brilliant red. Specimens can also be observed in this microscope by treating the specimens with fluorescent dyes like acridine orange R, auramine O, primulin, thiazo - yellow G etc. These fluorescent dye molecules are called as fluorochromes.
In practice, the microorganisms are stained with a fluorescent dye and the illuminated with blue light. The dye absorbs the blue light and emit green light.The fluorescence microscope consists of special components like exciter filter and barrier filter for the purpose. The function of the exciter filter is to remove all but the blue light. The barrier filter functions in blocking the blue light and allowing green light to pass through and reach eye. The selection of barrier filters depends on the dye used in microscopy.
The best adoption of this microscopy is Immuno-fluorescence’. Antibodies can be chemically combined with fluorescent dyes such as fluorescein isothiocyanate and lissamine rhodamine B. The antibodies. these labelled antibodies when mixed with a suspension of bacteria gets attached to the surface of the bacteria. These bacteria with attached labelled antibodies are clearly visible when observed by fluorescent microscopy. This procedure is known as fluorescent antibody technique and thephenomenon is called as immunofluorescence.
The fluorescence microscope is an essential and useful tool in medical microbiology and microbial ecology. Bacterial pathogens like Mycobacterium tuberculosis are easily identified by suing fluorochromes or fluorescent antibody technique.
Inverted Microscopes
There are two basic types of microscopes. The one most people are familiar with looks down at the specimen with the light source coming from below and is called an upright microscope. An inverted microscope looks up at the specimen with the light source coming from above instead.
Inverted microscopes were first invented in 1850 by Tulane University's J. Lawrence Smith and debuted at the World's Fair in London in 1852. In the early 20th century, they began to be used for observation of living cells, particularly for aquatic life. It was also used for analysis of heavy metals like iron and steel before World War II.
As the name suggests, an inverted microscope is upside down compared to a conventional microscope. The light source and condenser are on the top above the stage pointing down. The objectives and turret are below the stage pointing up.
The only things that are "standard" are that
1) a specimen (as dictated by the laws of gravity) is placed on top of the stage and
2) thank heavens, the binocular or trinocular tube is not upside down but in the standard position pointing at a conventional viewing angle. As a result, one is looking up through the bottom of whatever is holding the specimen and is sitting on the stage rather than looking at the specimen from the top, typically through a cover glass, as on a conventional microscope.
Grades of inverted microscope :
There are two grades of inverted microscopes.
1. A routine inverted microscope is small and comes in low and medium power settings. These can be used in homes and small labs in schools. They are limited in what the can observe as they usually do not allow for fine focus and have relatively low power magnification.
2. A research inverted microscope comes in heavy power settings and can allow for a very fine focus. The major disadvantage to them is that they are extremely expensive and are usually only used by universities and medical institutions. They are usually able to accommodate video cameras and televisions to assist in research documentation. The improvements on the inverted microscope over the course of the 20th and 21st centuries have allowed it to be an integral part of advanced scientific research.
Advantages :
1. An inverted microscope is most helpful when looking at heavy objects or those which are greatly effected by gravity. Material specimens like metal can be large and heavy. They require the large staging areas that inverted microscopes allow for.
2. The materials greatly affected by gravity include living cells and aquatic life that tend to pool and collect on the bottom of specimen containers. An inverted microscope looks at the sample from the bottom, making it easier to see the organisms with ease. It also allows users to see the samples in a more natural environment than a standard glass slide. Petri dishes allow more movement for the samples and are commonly used with inverted microscopes.
3. This type of microscope has been redesigned and improved on to accommodate particular uses. There are stages made particularly for processes like incubation and in vitro fertilization. The nosepieces have been made larger and revolvable, making to make it easier for scientists to identify and rotate objects. They have also been made heavier and sturdier, allowing for less vibration and greater ease of observation.
4. Inverted microscopes are often used for looking at living organisms and tissue that may be killed by staining, they often provide for "optical staining" through the use of phase contrast or DIC. My CK2 has phase contrast. Rather than use the more sophisticated phase contrast condenser used on a standard microscope, there is a simple slider that goes through the condenser and holds the necessary phase rings. Only the phase rings for the lower power objectives (e.g. 10x and 20x) are centerable by a fairly crude process (although it works). The phase ring for the 40x objective is not but seems to work fine.
4.1.2 Electron Microscope
Electron microscopes are of more recent origin and more sophisticated and useful than high microscopes. For the first time, Knoll and Rusca developed the electron microscope in 1932. The electron microscopy markedly differs in many respects from the optical microscopic technique. Electron microscope uses a beam of electrons and magnetic field as light and optical lenses, respectively, and the whole system operates in high vacuum. The electron microscope provide tremendous useful magnifications, because of much high resolution obtained than in light microscope.
As the electron beam has extremely short wavelength of only 0.05 A it provides much greater resolving power. The resolving power of the electron microscope is more than 100 times that of the light microscope, and it produces useful magnification up to x 40,000. There are two types of electron microscopes namely.
1. Transmission electron microscope ( TEM)
2. Scanning electron microscope (SEM)
1. Transmission electron microscope ( TEM) : The modern Transmission electron microscope is a complex and sophisticated microscope. The source of illumination in TEM is the electron gun which is made up of by a thin, V- shaped tungsten filament. On heating, the tungsten filament generates a beam electrons which is then focused on the specimen by the condenser. The specimen scatters the electrons passing through it. Then the beam is focused by objective magnet lens and project magnet lens to form an enlarged, visible image of the specimen on fluorescent screen. The magnified image can also be recorded on a photographic plate by a camera built into the instrument. Some substances are denser absorbing more electrons than others.
Usually, the penetrating power of the electrons through solid matter is weak. So, the specimens are prepared as either thin films or thin sections. To increase the contrast of these thin preparations of the biological specimens, they are usual treated with special electron microscopic stains. The important stains are osmic acid, permanganate, uranium, lanthanum and lead.
Specimen preparation : In order to observe specimens such as isolated macromolecules of protein or nucleic acid or to study the structural features of cells there are special techniques for specimen preparation. Two important techniques for specimen preparations are shadow casting and freeze etching.
i) Shadow casting technique : In this, the dried specimen is deposited with electron dense or heavy metal at an angle of about 45 from horizontal. The heavy metals normally used for this purpose are platinum, chromium, nickel, uranium and alloys of gold and platinum or platinum and palladium. This shadow casting technique increases the specimens contrast. This technique is particularly useful in studying virus morphology, bacterial flagella.
ii) Freeze etching technique : This is the more recent technique which avoids the fixation, embedding and sectioning of specimens. In this technique, the specimen is rapidly frozen in liquid nitrogen and then fractured with a knife pre-cooled with liquid nitrogen. The exposed surfaces of the fractured specimen are shadowed and coated with layers of platinum and carbon to form a replica of the surface. Later, these carbon replicas of the surfaces are used to study the details through TEM. This technique is found more useful in studying cell wall and membrane structures.
Draw backs of TEM :
1. Material to be observed should be dehydrated as TEM operates in vacuum.
2. The dehydration and treatment of specimen with electron dense staining substances may bring about changes in original structure.
3. The specimen preparation should not be thick, as electrons cannot penetrate through dense or thick material.
2. Scanning electron microscope (SEM) : The scanning electron microscope is a comparatively new type of electron microscope. It is quite different in principle and application from transmission electron microscope. The SEM gives a three dimensional quality to specimen images. It is useful in studying the surfaces of microorganisms in great detail. It possess a great depth of focus and can be used to observe even fairly large specimens.
- With this scanning electron microscope a wide range of magnifications from a low as X 15 up to about 1,00,000 is possible. In this a narrow beam of electrons rapidly move back and forth across the surface of metal coated specimen and scan the surface features. In contrast to the transmission electron microscope, the electrons emitted by the object’s surface produce an image in SEM.
The SEM consists a detector, photomultiplier, cathode ray tubes for, viewing and photography as special components. In scanning electron microscopy, the specimen preparation is comparatively simple and, the material to be observed is coated with a thin layer of heavy metal such as gold. In SEM, the beam of electrons released from the electronic gun strike the specimen and scans the surface. During this scanning, secondary electrons are released from the specimen. These secondary electrons are collected by a detector where an electronic signal is generated. These signals are amplified by photomultiplier and then sent to a cathode ray rube which produces an image like in a television system. This image can be viewed and photographed. The SEM is especially useful in studies, of bacterial cells, spores, fungi and morphological changes in tissues infected with microorganisms.
Draw backs of SEM :
1. The resolving power of SEM is comparatively less than that of transmission electron microscope.
2. It gives only the surface characters of the specimen but not of internal components.
The Cell
The cell
The first compound microscope was biolt by Robert Hooke, who used the term 'cell' in 1665 to describe the hollow spaces bound by cork in thin slices of cork. Thus started a new branch of biology - the study of the cell (cytology). In 1838 the German worker M.J. Schleiden discovered that all tissues of plants are made up of cells. In the same year, another German worker T.S Schwann arrived at teh same comclusion for animals. This concept was then applied to all living organisms. The findings of Schleiden and Schwann that all living organisms consist of cells are now referred to as the cell theory or the cell principle. The statements of Schleiden and Schwann have been slightly modified, and it is now held that living organisms consist of cells and cell products.
In the present section only a brief outline of the structure cell will be given. This will serve as an introduction to the organelles present in a generalised cell. The details of the structure and function of the organelles will be taken up in subsequent chapters.
Shape : The shape of the cell may be variable, i.e. constantly changing (e.g. Amoeba and leucocytes) or Fixed. In the latter case the cell may be : (i) flattened , e.g, squamous epethelium, endothelium and the upper layers of the epidermis; (ii) cuboidal, e.g. in thyroid gland fellicles; (iii) columnar, e.g. the cells lining the intestine; (iv) doscoidal e.g. erythrocytes, (v) spherical, e.g. the eggs of many animals; (vi) spindle-shaped, e.g. smooth muscle fibres; (vii) elongated, e.g. nerve cells, or (viii) branched, e.g. pigment cells of the skn.
Size: The size of cells vary from the very small cells of bacteria (0.2 to 5.0 microns) to the very large egg of the ostrich (6"). In the latter a considerabel part of the volume is made up of yolk, which is not protoplasm. Some nerve cells have axons as much as a metre in length.
The factors governing the size of the cell are : (i) the nucleocytoplasmic ratio, or the ratio between the volume of the nucleus and the cytoplasm, (ii) the ratio of the cell surface to the cell volume, and (iii) th rate of metabolism.
Protoplasm and Deutoplasm : The living substance of which the cell is made is called protoplasm. Protoplasm is differentiated into two regions, nucleoplasm and cytoplasm. Nucleoplasm is the protoplasm of sthe nucleus and cytoplasm the extra-nuclear protoplasm. The protoplasm of the cell contains many non-living substances which are mostly formed by the cell. These substances are collectively called deutoplasm, and include yolk bodies, lipid droplets, secretory granules and pigment. The cytoplasm may be differentiated into a granular peripheral region called ectoplasm (plasmagel, cortex) and a granular central region called endoplasm (medulla).
The cytoplasmic structures include the plasma membrane, the endoplasmic reticulum, the ribosimes, the Golgi complex, the mitochondria, the chloroplasts (in plant cells), the centrioles,the lysosomes, the cilia, the flagella and the vacuoles. The nucleoplasm consists of the nuclear envelope, nuclear sap,chromatin, chromosomes and nucleoli.
Plasma membrane : The cell is bounded by a lipoprotein membrane which is selectively permeable, and in specialized cells like nerve cells is responsible for the conduction of impulses (excitation). The plasmamembrane together with the surrounding cell cement forms the cell membrane, the structure visible under the light microscope. In plant cells the cell membrane is surrounded by a cell wall.
The Golgi complex consists of a system of smooth membranes in the form of cisternae and besicles. Various functins have been ascribed to the Golgi complex. The main function is the concentration and budding off of secretory products ans carbohydrate synthesis.
Scattered through the cytoplasm are granular or rod-like bodies called mitochondria. They contain the enzymesfor the Krebs cycle and oxidative phosphorylation. Mitochondria have been called the powerhouses of the cell and are the centres of cell respiration and release of enrgy.
Chloroplasts are chlorophyll-containing bodies fornd in plant cells. They contain structure called quantosomes which are the units of photosynthesis.
Lysosomes are particles consisting of hydrolytic enzymes enclosed within lipoprotein membranes. The lysosomes have been called 'suicide bags' because the enzymes cause breakdown and death of the cell. Lysosomes are concerned with extra-cellular and intra-cellular digestion, cell breakdown, penetration of the sperm, and initiation of cell division.
The cytoplasm contains vacuoles, which are fluid-filled spaces enclosed by membranes. They are well developed in plant cells, but absent from most animal cells, except the Protozoa.
In both plants and animals some3 cells have hair-like structures called cilia and flagella. At the base of each cilium and flagellum is a basal body or kinetosome.
Eukaryote cells have nuclei which are bounded by a double membraned nuclear envelope perforated by pores. The nucleus is filled with a colourless fluid called nuclear sap. Within the sap is a network of chromatin. The nucleus may contain one of more nucleoli which are the storage sites of nuclear RNA. During cell division, filamentous bodies called chromosomes appear in the nucleus. The chromisomes contain DNA which acts as a template for the synthesis of RNA.
PROKARYOTE AND EUKARYOTE CELLS
Organisms in which the nuclear material is not bounded by a definite nuclear membrane are called prokaryotes, e.g. bacteria and blue-green algae (now included in bacteria). Organisms in which the nucleus has a definite nuclear membrane are known as eukaryotes, e.g. all other plants and animals. The cells of prokaryotes and eukaryotes differ fundamentally in many ways.
1. Nuclear membrane : Prokaryotic cells lack a nuclear membrane while eukaryotic cellshave a definite nuclear membrane.
2. Chromosomes : In prokaryotes the genetic material consists of nucleic acid (DNA). The DNA molecule is circular and lies in a tangled mass (nucleoid). In eukaryotes the nucleic acid (DNA) is associated with proteins to form definite bodies called chromosomes.
3. Cytoplamic organelles : Prokaryotic cells have no endoplasmic reticulum, Golgi complex, mitochondria, lysosomes or centrioles. The enzymatic functions of the mitochondria are carried out by the cell membrane, which is folded inwards at various points. Eukaryotic cells have definite internal membraneous structures like the endoplasmic reticulum, Golgi complex, mitochondria and lysosomes.
4. Cell wall : The cell wall of prokaryotes contains amino sugars and muramic acid. In eukaryotes the cell wall, when present, does not contain these substances.
5. Flagella : The flagella and cilia of eukaryotes have a definite structure consisting of 2 central and 9 peripheral fibrils. Some prokayotes have flagella, but theflagella do not have the 9+2 internal structure.
6. Cytoplasmic streaming or amoeboid movememt may occur in eukaryotic cells but does not occur in prokaryotes.
7. Photosynthetic apparatus : In prokaryotes chlorophyll, when present, is associated with lamellae. These lamellae are, however, not enclosed by membranes and hence no distinct choloroplasts are present. In eukaryotes, chlorophyll, when present, is found in chlorophasts.
PROTOPLASM
Protoplasm has been defined as the 'material basis of life'. The term'protoplam' was coined in 1839 by the Bohemian physoilogist Johannes Purkinje. Protoplasm refers to the substance of which the cell is made and includes all parts of teh cell. It sis considered to be a living substance, since it metabolizes and self-perpetuates. The term deutoplasm is used to describe the substances formed by the protoplasm. Protoplasm is divided into nucleoplasm or protoplasm of the nucleus and cytoplasm or extra-nuclear protoplasm.
Protoplasm is made up mainly of oxygen, carbon, hydrogen and nitrogen, which make up 95% by weight of the body. It also has a group of minerals. Protoplasm is made up of both inorganic and organic substances. Water, the main inorganic substance, varies from 5% to 90% in different tissues, with an average of 65% to 75%.
ORGANIZATION AND DIVERSITY OF CELLS
All organisms more complex than viruses consist of cells, aqueous compartments bounded by membranes, which under restricted conditions are capable of existing independently. All cells are derived by cell division from other cells. All ellular organisms can be subdivided into two major classes, prokaryotes and eukaryotes, on the basis of the architecture of their cells.
Prokaryotes lack a defined nucleus and have a relatively simple internal organization. Under the electron microscope they appear relatively featureless. They comprise two kingdoms of life : eubacteria which include most of the bacteria ; and the archaea, rather poorly understood organisms that superficially resemble bacteria and often grow in unusual environments, such as in acid hot springs, saturated brines, etc. Eukaryotes are thought to have first appeared about 1.5 billion years ago. There is only one kingdom of eukaryotic organisms - the eukarya - but this includes Protists Fungi, Animals and Plants. Eukaryotes have a much more complex intracellular organization with internal membranes, membrane-bound organelles including a nucleus, and a well-organized cytoskeleton as compared to prokaryotes.
1.1.2 VIRAL CELL
Viruses are simple, noncellular entities consisting of one or more molecules of either DNA or RNA enclosed in a coat of protein, They can reprodeuce only within living cells and are obligate intracellular parasites. Viruses are smaller than prokaryotic cells ranging in size from 0.02 to 0.3 m (small pox virus is largest virus about 200 nm in diameter and polio virus is the smallest virus about 28 nm in diameter).
A fully assembled infectious virus is called a virion. The main function of the virion is to deliver its DNA or RNA genome into the host cell so that the genome can be expressed (transcribed and translated) by the host cell. Each viral species has a very limited host range; i.e., it can reproduce in only a small group of closely related species.
Viral structure : The structure of virions are very deverse, varying widely in size, shape and chemical composition. All viruses have a nucleocapsid composed of nucleic acid surrounded by a protein capsid. A protein coat, the capsid, which functions as a shell to protect the viral genome from nucleases and which during infection attaches the virion to specific receptors exposed on the prospective host cell. Capsids are formed as single or double protein shells and consists of only one or a few structural protein species. The proteins used to build the capsid are called protomers. The nucleic acid-associated protein, called nucleoprotein, together with the genome, forms the nucleocapsid. Some viruses have a membranous envelope that lies outside the nucleocapsid. Those virions having an envelope are called enveloped viruses; where as those lacking a envelope are called naked viruses. In enveloped viruses, the nucleocapsid is surrounded by a lipid bilayer and glycoprotein derived from the modified host cell membrane. Enveloped viruses often exhibit a fringe of glycoprotein spikes or knobs, also called peplomers. In viruses that acquire their envelope by budding through the plasma membrane or another intracellular cell membrane, the lipid composition of the viral envelope closely reflects that of the particular host membrane.
- Viral genomes are smaller in size. The largest known viral genome, that of bacteriophage G, is 670 kbs. The genome of a virus may consist of DNA or RNA, which may be single stranded (ss) or double stranded (ds), linear or circular. The entire genome may occupy either one nucleic acid molecule (monopartite genome) or several RNA viruses is called sense (positive sense, plus sense) in orientation of it can serve as mRNA, and antisense (negative sense, minus sense) if a complememtary strand synthesized by a viral RNA transcriptase serves as mRNA.
Shape/symmetry : All viruses have a nucleocapsid (nucleic acid and protein) structure. The symmetry ( refers to the way in which the capsomeres are arranged in the virus capsid) may be icosahedral ( spherical shape) or helical (rod shape).
Helical symmetry : It is seen in nucleocapsids of many filamentous and pleomorphic viruses. Helical nucleocapsids consist of a helical array of capsid proteins (protomers) wrapped around a helical filament of nucleic acid. A typical virus with helical symmetry is TMV.
Icosahedral morphology : It is characteristic of the nucleocapsids of many 'spherical' viruses. An icosahedron is a regular polyhedron with 20 equilateral triangular faces and 12 vertices. Complex structures have capsid symmetry that is neither purely icosahedral nor helical. As for example T4 virus of E.coli.
BACTERIOPHAGES (BACTERIAL VIRUS)
Phages were first observed in 1915 by F. Twort in England and in 1917 by F. d'Herelle in France. D'Herelle named them bacteriophage (eaters of bacteria). A bacteriophage is a virus that infects bacteria and sometimes destroys them by lysis, or dissolution of the cell. Bacteriophages, or phages, have a head composed of protein, an inner core of nucleic acid - either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and a hollow protein tail. A particular phage can usually infect only one or a few related species of bacteria ; for example, coliphages are DNA- containing viruses that infect only the bacterium Escherichia coli.
Several morphologically distrinct types of phage have been described, including polyhedral, filamentous, and complex. Complex phages have polyhedral heads to which tails and sometimes other appendages (tail plates, tail fibers, etc.) are attached.
EUKATYOTIC VIRUSES
Animal Viruses : Animal viruses have a variety of shapes, sizes, and nature of genome. The genome of animal of animal viruses may be DNA or RNA, ss or ds, linear or circular, segmented or non-segmented.
Retroviruses : Retroviruses are ssRNA (plus sense) containing animal vurus that replicates through a DNA intermediates. Retroviruses are enveloped viruses. The enzymes found in the virus particle are reverse transcriptase, integrase and a protease. Ex : HIV-I
Plant Viruses : Plant viruses exist in rod and polyhedral shape. Most plant viruses have genomes consisting of a single RNA strand of the (+) type. The best-known plant virus is the rod-shaped tobacco mosaic virus (TMV).
Prions : Virus without Nucleic Acid is called as Prions. Prions are proteinaceons infectious agents that are responsible for neurodegenerative diseases in animals including human.
Viroid : Virus without Protein is called as Viroid. Viroids have so far been shown to infect plants only. Virusoids are satellite nucleic acids.
1.1.3 BACTERIAL CELL
Bacteria are microscpic, relatively simple, prokaryotic organisms whose cells lack a nucleus or nuclear membrane. Bacteria vary in size from less than 0.2m in diameter to more than 50 micro meters in diameter. The largest known bacterium in terms of total cells volume is Thiomargarita namibiensis, whereas smallest known example is Mycoplasma pneumoniae. The bacteria may appear as rod (bacilli), sphere (cocci), or spiral (spirilla or spirochetes) shaped. Bacteria reproduce by binary fission, have unique cell walls, and exist in most environments on earth. Thay live at temperatures ranging from 0oC to over 100oC and in conditions that are oxygen-rich or oxygen-free.
Structure : Cell Wall : The cell wall of both Gram-positive and Gram-negative bacteria is chemically peptidoglycans (murein), which confer the characteristic cell shape. Peptidoglycans are unique to prokaryotic organisms. In gram-positive bacteria peptidoglycans cell wall consists of a single 20 to 80 nm thick homogeneous layer lying outside the plasma membrane. In contrast, the gram-negative cell wall consists of 2 to 7 nm thick peptidoglycan layer covered by a to 8 nm thick outer membrane. The space that is present between the plasma membrane and the outer membrane in gram-negative bacteria is called the periplasmic space. The substance that occupies the periplasmic space is called periplasm. Periplasmic space is also present in some gram-positive bacteria.
Peptidoglycan is a polymer contains two sugar derivatives N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) joined through -1,4 glycosidic bond. A peptide chain of four alternating D- and L- amino acids called tetrapeptide is connected to the carboxyl group of the NAM.
Many gram-positive bacteria have acidic substances called teichoic acids. Teichoic acids are polyol phosphate polymers bearing a strong negative charge. They are covalently linked to the peptidoglycan. They are strongly antigenic, but are generally absent in Gram-negative bacteria.
Capsules : Some bacteria form a thick high-molecular weight, viscous polysaccharide material on their outer surface. These thick walled cells are called as capsules. The terms capsule and slime layer are frequently used to describe these layers.
Plasma membrane : The bacterial plasma membrane is an unit membrane composed primarily of protein and phospholipid. One major difference in chemical composition of membranes between eukaryotic and prokaryotic cells is that eukaryotes have sterols in their membranes and sterols are absent in prokaryotic cell membranes except in Mycoplasma. An invagination or infolding of the plama membrane (called mesosome) is present in bacteria that may be invilved in chromosome replication or cross wall formation in dividing bacteria.
Cytoplasm : The bacterial cytoplasm is densely packed with 70S ribosomes. Bacterial cytoplasm also contains many organic and inorganic granules that may be non-unit membrane bound or membraneless. These granules may be Glycogen, Poly--hydroxy butyrate (PHB), Cyanophycin granules (composed of large polypeptides containing approximately equal amounts of amino acids arginine and aspartic acid; present in cyanobacteria), Carboxysomes (polyhedral inclusion bodies that contain the CO2 fixation enzymes ribules-1,5-bisphosphate carboxylase; found in cyanobacteria, nitrifying bacteria, and thiobacilli) and metachromatic granules (granules of polyphosphate in the cytoplasm of some bacteria that show differences in colour when stained with a blue basic dye; acts as reserve of phosphate).
Surface appendages : Two types of surface appendage can be recognized on certain bacterial species ;
i. Flagella : Flagella occur in both Gram-positive and Gram-negative bacteria. These are used in locomotion of bacteria. Structurally, bacterial flagella are long, filamentous surface appendages. A flagellum consists of three parts; Filament, hook, basal body.
ii. Pili : Pili occur almost exclusively on Gram-negative bacteria and are found on only a few Gram-positive organisms (e.g., Corylebacterium renale). The terms pili and fimbriae are usually used interchangeably to describe the thin, hairlike appendages on the surface of many Gram-negative bacteria. Proteins that form pili are referred to as pilins.
Endospores : Bacillus and Clostridium species can produce endospores; heat-resistant, dehydrated resting cells that are formed intracellularly and contain a genome and all essential metabolic machinery. Endospore structure is very complex and has many layers that are absent from the vegetative cell.
1.1.4 FUNGAL CELL
Fungal cells are typically eukaryotic but lack chloroplasts. They contain most of the familiar organelles characteristic to eukaryotes. The ultra structural details of a fungal cell can be studied under the following four main structural components : 1. cell wall, 2. plasma membrane (plasmalemma) and its invaginations, 3. organelles, and 4. inclusions in cytoplasm.
The cell wall of most fungi contain chitin, a polymer of -1,4 linked 2-acetamido-2-deoxy-D-glucopyranose. Chitin and cellulose are found together as in Rhizidiomyces and in Ceratocystis. Various other substances have been found associated together with chitin and cellulose in fungal cell walls.
Plasma membrane is a unit membrane which, like other eukaryotes, shows the fluid mosaic structure. Some exciting developments in the ultrastructure of fungal cells have been found. Invaginations of plasma membrane, similar to yeasts have also been observed in Neurospora crassa and Schizophyllum commune.
The various types of organelles that occur in fungal cells are the following.
1. Nucleus : Fungal nuclei are small, rounded bodies, 2-3 m in diameter but occasionally 30m in diameter. Nuclei are bounded by a double nucleare membrane, continuous with ER. There are numerous pores in the nuclear envelope.
2. Mitochondria : Mitochondria are remarkably pleomorphic. They are of diverse shapes, ranging from small, spherical structures capable of elongating to 30m in length t unequally thickened structures resembling a row of beads on a thread, and may even branch.
3. Microbodies : Microbodies are widespread in fungi and described as single unit membranes surrounding an amorphous, crystalline or fibrillar matrix. They are oval or round in shape, 0.5-1.5 m in diameter.
4. Ribosomes : Ribosomes are rich in protein and RNA and occur both in the cytoplasm and mitochondria. Cytoplasmic ribosomes are more or less spherical and 15-25 nm in diameter. Polyribosomes or polysomes (aggregation of ribosomes) have also been detected. They may be bound with ER.
5.Vacuoles, Lysosomes and Vesicles : Vacuoles are first visible in subapical regions of hyphae. There are many vacuoles in yeast cells. They accumulate pigments, aminoacids, and hydrolases.
Lysosomes have been identified in several fungi. They are single -membrane bound organelles, about 400nm in diameter containing more than one hydrolytic enzyme with an acidic pH optimum.
Vesicles are common in fungi, especially in the apical region and wherever wall synthesis is in progress.
6. Microtubules : Microtubules have been identified during nuclear division, where they not only develop within the nuclear membrane but also radiate out beyond the poles into the cytoplasm.
1.1.5 ANIMAL AND PLANT CELLS
Plant and animal cells are structurally very similar because they are both eukaryotic cells.
Similarities : They both contain membrane-bound organelles such as the nucleus, mitochondria, endoplasmic reticulum, golgi apparatus, lysosomes, and peroxisomes. Both are contain similar membranes, cytosol, and cytoskeletal elements.
Differences : Plant cells can be larger than animal cells. the normal range for an animal cell varies from 10 to 30 m while that for a plant cell stretches from 10 to 100 m. Beyond size, the main structural differences between plant and animal cells lie in a few additional structures found in animal cells. These structures include ; chloroplast, the cell wall, and vacuoles.
A detailed description of eukaryotic cells is provided in later chapters.
The first compound microscope was biolt by Robert Hooke, who used the term 'cell' in 1665 to describe the hollow spaces bound by cork in thin slices of cork. Thus started a new branch of biology - the study of the cell (cytology). In 1838 the German worker M.J. Schleiden discovered that all tissues of plants are made up of cells. In the same year, another German worker T.S Schwann arrived at teh same comclusion for animals. This concept was then applied to all living organisms. The findings of Schleiden and Schwann that all living organisms consist of cells are now referred to as the cell theory or the cell principle. The statements of Schleiden and Schwann have been slightly modified, and it is now held that living organisms consist of cells and cell products.
In the present section only a brief outline of the structure cell will be given. This will serve as an introduction to the organelles present in a generalised cell. The details of the structure and function of the organelles will be taken up in subsequent chapters.
Shape : The shape of the cell may be variable, i.e. constantly changing (e.g. Amoeba and leucocytes) or Fixed. In the latter case the cell may be : (i) flattened , e.g, squamous epethelium, endothelium and the upper layers of the epidermis; (ii) cuboidal, e.g. in thyroid gland fellicles; (iii) columnar, e.g. the cells lining the intestine; (iv) doscoidal e.g. erythrocytes, (v) spherical, e.g. the eggs of many animals; (vi) spindle-shaped, e.g. smooth muscle fibres; (vii) elongated, e.g. nerve cells, or (viii) branched, e.g. pigment cells of the skn.
Size: The size of cells vary from the very small cells of bacteria (0.2 to 5.0 microns) to the very large egg of the ostrich (6"). In the latter a considerabel part of the volume is made up of yolk, which is not protoplasm. Some nerve cells have axons as much as a metre in length.
The factors governing the size of the cell are : (i) the nucleocytoplasmic ratio, or the ratio between the volume of the nucleus and the cytoplasm, (ii) the ratio of the cell surface to the cell volume, and (iii) th rate of metabolism.
Protoplasm and Deutoplasm : The living substance of which the cell is made is called protoplasm. Protoplasm is differentiated into two regions, nucleoplasm and cytoplasm. Nucleoplasm is the protoplasm of sthe nucleus and cytoplasm the extra-nuclear protoplasm. The protoplasm of the cell contains many non-living substances which are mostly formed by the cell. These substances are collectively called deutoplasm, and include yolk bodies, lipid droplets, secretory granules and pigment. The cytoplasm may be differentiated into a granular peripheral region called ectoplasm (plasmagel, cortex) and a granular central region called endoplasm (medulla).
The cytoplasmic structures include the plasma membrane, the endoplasmic reticulum, the ribosimes, the Golgi complex, the mitochondria, the chloroplasts (in plant cells), the centrioles,the lysosomes, the cilia, the flagella and the vacuoles. The nucleoplasm consists of the nuclear envelope, nuclear sap,chromatin, chromosomes and nucleoli.
Plasma membrane : The cell is bounded by a lipoprotein membrane which is selectively permeable, and in specialized cells like nerve cells is responsible for the conduction of impulses (excitation). The plasmamembrane together with the surrounding cell cement forms the cell membrane, the structure visible under the light microscope. In plant cells the cell membrane is surrounded by a cell wall.
The Golgi complex consists of a system of smooth membranes in the form of cisternae and besicles. Various functins have been ascribed to the Golgi complex. The main function is the concentration and budding off of secretory products ans carbohydrate synthesis.
Scattered through the cytoplasm are granular or rod-like bodies called mitochondria. They contain the enzymesfor the Krebs cycle and oxidative phosphorylation. Mitochondria have been called the powerhouses of the cell and are the centres of cell respiration and release of enrgy.
Chloroplasts are chlorophyll-containing bodies fornd in plant cells. They contain structure called quantosomes which are the units of photosynthesis.
Lysosomes are particles consisting of hydrolytic enzymes enclosed within lipoprotein membranes. The lysosomes have been called 'suicide bags' because the enzymes cause breakdown and death of the cell. Lysosomes are concerned with extra-cellular and intra-cellular digestion, cell breakdown, penetration of the sperm, and initiation of cell division.
The cytoplasm contains vacuoles, which are fluid-filled spaces enclosed by membranes. They are well developed in plant cells, but absent from most animal cells, except the Protozoa.
In both plants and animals some3 cells have hair-like structures called cilia and flagella. At the base of each cilium and flagellum is a basal body or kinetosome.
Eukaryote cells have nuclei which are bounded by a double membraned nuclear envelope perforated by pores. The nucleus is filled with a colourless fluid called nuclear sap. Within the sap is a network of chromatin. The nucleus may contain one of more nucleoli which are the storage sites of nuclear RNA. During cell division, filamentous bodies called chromosomes appear in the nucleus. The chromisomes contain DNA which acts as a template for the synthesis of RNA.
PROKARYOTE AND EUKARYOTE CELLS
Organisms in which the nuclear material is not bounded by a definite nuclear membrane are called prokaryotes, e.g. bacteria and blue-green algae (now included in bacteria). Organisms in which the nucleus has a definite nuclear membrane are known as eukaryotes, e.g. all other plants and animals. The cells of prokaryotes and eukaryotes differ fundamentally in many ways.
1. Nuclear membrane : Prokaryotic cells lack a nuclear membrane while eukaryotic cellshave a definite nuclear membrane.
2. Chromosomes : In prokaryotes the genetic material consists of nucleic acid (DNA). The DNA molecule is circular and lies in a tangled mass (nucleoid). In eukaryotes the nucleic acid (DNA) is associated with proteins to form definite bodies called chromosomes.
3. Cytoplamic organelles : Prokaryotic cells have no endoplasmic reticulum, Golgi complex, mitochondria, lysosomes or centrioles. The enzymatic functions of the mitochondria are carried out by the cell membrane, which is folded inwards at various points. Eukaryotic cells have definite internal membraneous structures like the endoplasmic reticulum, Golgi complex, mitochondria and lysosomes.
4. Cell wall : The cell wall of prokaryotes contains amino sugars and muramic acid. In eukaryotes the cell wall, when present, does not contain these substances.
5. Flagella : The flagella and cilia of eukaryotes have a definite structure consisting of 2 central and 9 peripheral fibrils. Some prokayotes have flagella, but theflagella do not have the 9+2 internal structure.
6. Cytoplasmic streaming or amoeboid movememt may occur in eukaryotic cells but does not occur in prokaryotes.
7. Photosynthetic apparatus : In prokaryotes chlorophyll, when present, is associated with lamellae. These lamellae are, however, not enclosed by membranes and hence no distinct choloroplasts are present. In eukaryotes, chlorophyll, when present, is found in chlorophasts.
PROTOPLASM
Protoplasm has been defined as the 'material basis of life'. The term'protoplam' was coined in 1839 by the Bohemian physoilogist Johannes Purkinje. Protoplasm refers to the substance of which the cell is made and includes all parts of teh cell. It sis considered to be a living substance, since it metabolizes and self-perpetuates. The term deutoplasm is used to describe the substances formed by the protoplasm. Protoplasm is divided into nucleoplasm or protoplasm of the nucleus and cytoplasm or extra-nuclear protoplasm.
Protoplasm is made up mainly of oxygen, carbon, hydrogen and nitrogen, which make up 95% by weight of the body. It also has a group of minerals. Protoplasm is made up of both inorganic and organic substances. Water, the main inorganic substance, varies from 5% to 90% in different tissues, with an average of 65% to 75%.
ORGANIZATION AND DIVERSITY OF CELLS
All organisms more complex than viruses consist of cells, aqueous compartments bounded by membranes, which under restricted conditions are capable of existing independently. All cells are derived by cell division from other cells. All ellular organisms can be subdivided into two major classes, prokaryotes and eukaryotes, on the basis of the architecture of their cells.
Prokaryotes lack a defined nucleus and have a relatively simple internal organization. Under the electron microscope they appear relatively featureless. They comprise two kingdoms of life : eubacteria which include most of the bacteria ; and the archaea, rather poorly understood organisms that superficially resemble bacteria and often grow in unusual environments, such as in acid hot springs, saturated brines, etc. Eukaryotes are thought to have first appeared about 1.5 billion years ago. There is only one kingdom of eukaryotic organisms - the eukarya - but this includes Protists Fungi, Animals and Plants. Eukaryotes have a much more complex intracellular organization with internal membranes, membrane-bound organelles including a nucleus, and a well-organized cytoskeleton as compared to prokaryotes.
1.1.2 VIRAL CELL
Viruses are simple, noncellular entities consisting of one or more molecules of either DNA or RNA enclosed in a coat of protein, They can reprodeuce only within living cells and are obligate intracellular parasites. Viruses are smaller than prokaryotic cells ranging in size from 0.02 to 0.3 m (small pox virus is largest virus about 200 nm in diameter and polio virus is the smallest virus about 28 nm in diameter).
A fully assembled infectious virus is called a virion. The main function of the virion is to deliver its DNA or RNA genome into the host cell so that the genome can be expressed (transcribed and translated) by the host cell. Each viral species has a very limited host range; i.e., it can reproduce in only a small group of closely related species.
Viral structure : The structure of virions are very deverse, varying widely in size, shape and chemical composition. All viruses have a nucleocapsid composed of nucleic acid surrounded by a protein capsid. A protein coat, the capsid, which functions as a shell to protect the viral genome from nucleases and which during infection attaches the virion to specific receptors exposed on the prospective host cell. Capsids are formed as single or double protein shells and consists of only one or a few structural protein species. The proteins used to build the capsid are called protomers. The nucleic acid-associated protein, called nucleoprotein, together with the genome, forms the nucleocapsid. Some viruses have a membranous envelope that lies outside the nucleocapsid. Those virions having an envelope are called enveloped viruses; where as those lacking a envelope are called naked viruses. In enveloped viruses, the nucleocapsid is surrounded by a lipid bilayer and glycoprotein derived from the modified host cell membrane. Enveloped viruses often exhibit a fringe of glycoprotein spikes or knobs, also called peplomers. In viruses that acquire their envelope by budding through the plasma membrane or another intracellular cell membrane, the lipid composition of the viral envelope closely reflects that of the particular host membrane.
- Viral genomes are smaller in size. The largest known viral genome, that of bacteriophage G, is 670 kbs. The genome of a virus may consist of DNA or RNA, which may be single stranded (ss) or double stranded (ds), linear or circular. The entire genome may occupy either one nucleic acid molecule (monopartite genome) or several RNA viruses is called sense (positive sense, plus sense) in orientation of it can serve as mRNA, and antisense (negative sense, minus sense) if a complememtary strand synthesized by a viral RNA transcriptase serves as mRNA.
Shape/symmetry : All viruses have a nucleocapsid (nucleic acid and protein) structure. The symmetry ( refers to the way in which the capsomeres are arranged in the virus capsid) may be icosahedral ( spherical shape) or helical (rod shape).
Helical symmetry : It is seen in nucleocapsids of many filamentous and pleomorphic viruses. Helical nucleocapsids consist of a helical array of capsid proteins (protomers) wrapped around a helical filament of nucleic acid. A typical virus with helical symmetry is TMV.
Icosahedral morphology : It is characteristic of the nucleocapsids of many 'spherical' viruses. An icosahedron is a regular polyhedron with 20 equilateral triangular faces and 12 vertices. Complex structures have capsid symmetry that is neither purely icosahedral nor helical. As for example T4 virus of E.coli.
BACTERIOPHAGES (BACTERIAL VIRUS)
Phages were first observed in 1915 by F. Twort in England and in 1917 by F. d'Herelle in France. D'Herelle named them bacteriophage (eaters of bacteria). A bacteriophage is a virus that infects bacteria and sometimes destroys them by lysis, or dissolution of the cell. Bacteriophages, or phages, have a head composed of protein, an inner core of nucleic acid - either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and a hollow protein tail. A particular phage can usually infect only one or a few related species of bacteria ; for example, coliphages are DNA- containing viruses that infect only the bacterium Escherichia coli.
Several morphologically distrinct types of phage have been described, including polyhedral, filamentous, and complex. Complex phages have polyhedral heads to which tails and sometimes other appendages (tail plates, tail fibers, etc.) are attached.
EUKATYOTIC VIRUSES
Animal Viruses : Animal viruses have a variety of shapes, sizes, and nature of genome. The genome of animal of animal viruses may be DNA or RNA, ss or ds, linear or circular, segmented or non-segmented.
Retroviruses : Retroviruses are ssRNA (plus sense) containing animal vurus that replicates through a DNA intermediates. Retroviruses are enveloped viruses. The enzymes found in the virus particle are reverse transcriptase, integrase and a protease. Ex : HIV-I
Plant Viruses : Plant viruses exist in rod and polyhedral shape. Most plant viruses have genomes consisting of a single RNA strand of the (+) type. The best-known plant virus is the rod-shaped tobacco mosaic virus (TMV).
Prions : Virus without Nucleic Acid is called as Prions. Prions are proteinaceons infectious agents that are responsible for neurodegenerative diseases in animals including human.
Viroid : Virus without Protein is called as Viroid. Viroids have so far been shown to infect plants only. Virusoids are satellite nucleic acids.
1.1.3 BACTERIAL CELL
Bacteria are microscpic, relatively simple, prokaryotic organisms whose cells lack a nucleus or nuclear membrane. Bacteria vary in size from less than 0.2m in diameter to more than 50 micro meters in diameter. The largest known bacterium in terms of total cells volume is Thiomargarita namibiensis, whereas smallest known example is Mycoplasma pneumoniae. The bacteria may appear as rod (bacilli), sphere (cocci), or spiral (spirilla or spirochetes) shaped. Bacteria reproduce by binary fission, have unique cell walls, and exist in most environments on earth. Thay live at temperatures ranging from 0oC to over 100oC and in conditions that are oxygen-rich or oxygen-free.
Structure : Cell Wall : The cell wall of both Gram-positive and Gram-negative bacteria is chemically peptidoglycans (murein), which confer the characteristic cell shape. Peptidoglycans are unique to prokaryotic organisms. In gram-positive bacteria peptidoglycans cell wall consists of a single 20 to 80 nm thick homogeneous layer lying outside the plasma membrane. In contrast, the gram-negative cell wall consists of 2 to 7 nm thick peptidoglycan layer covered by a to 8 nm thick outer membrane. The space that is present between the plasma membrane and the outer membrane in gram-negative bacteria is called the periplasmic space. The substance that occupies the periplasmic space is called periplasm. Periplasmic space is also present in some gram-positive bacteria.
Peptidoglycan is a polymer contains two sugar derivatives N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) joined through -1,4 glycosidic bond. A peptide chain of four alternating D- and L- amino acids called tetrapeptide is connected to the carboxyl group of the NAM.
Many gram-positive bacteria have acidic substances called teichoic acids. Teichoic acids are polyol phosphate polymers bearing a strong negative charge. They are covalently linked to the peptidoglycan. They are strongly antigenic, but are generally absent in Gram-negative bacteria.
Capsules : Some bacteria form a thick high-molecular weight, viscous polysaccharide material on their outer surface. These thick walled cells are called as capsules. The terms capsule and slime layer are frequently used to describe these layers.
Plasma membrane : The bacterial plasma membrane is an unit membrane composed primarily of protein and phospholipid. One major difference in chemical composition of membranes between eukaryotic and prokaryotic cells is that eukaryotes have sterols in their membranes and sterols are absent in prokaryotic cell membranes except in Mycoplasma. An invagination or infolding of the plama membrane (called mesosome) is present in bacteria that may be invilved in chromosome replication or cross wall formation in dividing bacteria.
Cytoplasm : The bacterial cytoplasm is densely packed with 70S ribosomes. Bacterial cytoplasm also contains many organic and inorganic granules that may be non-unit membrane bound or membraneless. These granules may be Glycogen, Poly--hydroxy butyrate (PHB), Cyanophycin granules (composed of large polypeptides containing approximately equal amounts of amino acids arginine and aspartic acid; present in cyanobacteria), Carboxysomes (polyhedral inclusion bodies that contain the CO2 fixation enzymes ribules-1,5-bisphosphate carboxylase; found in cyanobacteria, nitrifying bacteria, and thiobacilli) and metachromatic granules (granules of polyphosphate in the cytoplasm of some bacteria that show differences in colour when stained with a blue basic dye; acts as reserve of phosphate).
Surface appendages : Two types of surface appendage can be recognized on certain bacterial species ;
i. Flagella : Flagella occur in both Gram-positive and Gram-negative bacteria. These are used in locomotion of bacteria. Structurally, bacterial flagella are long, filamentous surface appendages. A flagellum consists of three parts; Filament, hook, basal body.
ii. Pili : Pili occur almost exclusively on Gram-negative bacteria and are found on only a few Gram-positive organisms (e.g., Corylebacterium renale). The terms pili and fimbriae are usually used interchangeably to describe the thin, hairlike appendages on the surface of many Gram-negative bacteria. Proteins that form pili are referred to as pilins.
Endospores : Bacillus and Clostridium species can produce endospores; heat-resistant, dehydrated resting cells that are formed intracellularly and contain a genome and all essential metabolic machinery. Endospore structure is very complex and has many layers that are absent from the vegetative cell.
1.1.4 FUNGAL CELL
Fungal cells are typically eukaryotic but lack chloroplasts. They contain most of the familiar organelles characteristic to eukaryotes. The ultra structural details of a fungal cell can be studied under the following four main structural components : 1. cell wall, 2. plasma membrane (plasmalemma) and its invaginations, 3. organelles, and 4. inclusions in cytoplasm.
The cell wall of most fungi contain chitin, a polymer of -1,4 linked 2-acetamido-2-deoxy-D-glucopyranose. Chitin and cellulose are found together as in Rhizidiomyces and in Ceratocystis. Various other substances have been found associated together with chitin and cellulose in fungal cell walls.
Plasma membrane is a unit membrane which, like other eukaryotes, shows the fluid mosaic structure. Some exciting developments in the ultrastructure of fungal cells have been found. Invaginations of plasma membrane, similar to yeasts have also been observed in Neurospora crassa and Schizophyllum commune.
The various types of organelles that occur in fungal cells are the following.
1. Nucleus : Fungal nuclei are small, rounded bodies, 2-3 m in diameter but occasionally 30m in diameter. Nuclei are bounded by a double nucleare membrane, continuous with ER. There are numerous pores in the nuclear envelope.
2. Mitochondria : Mitochondria are remarkably pleomorphic. They are of diverse shapes, ranging from small, spherical structures capable of elongating to 30m in length t unequally thickened structures resembling a row of beads on a thread, and may even branch.
3. Microbodies : Microbodies are widespread in fungi and described as single unit membranes surrounding an amorphous, crystalline or fibrillar matrix. They are oval or round in shape, 0.5-1.5 m in diameter.
4. Ribosomes : Ribosomes are rich in protein and RNA and occur both in the cytoplasm and mitochondria. Cytoplasmic ribosomes are more or less spherical and 15-25 nm in diameter. Polyribosomes or polysomes (aggregation of ribosomes) have also been detected. They may be bound with ER.
5.Vacuoles, Lysosomes and Vesicles : Vacuoles are first visible in subapical regions of hyphae. There are many vacuoles in yeast cells. They accumulate pigments, aminoacids, and hydrolases.
Lysosomes have been identified in several fungi. They are single -membrane bound organelles, about 400nm in diameter containing more than one hydrolytic enzyme with an acidic pH optimum.
Vesicles are common in fungi, especially in the apical region and wherever wall synthesis is in progress.
6. Microtubules : Microtubules have been identified during nuclear division, where they not only develop within the nuclear membrane but also radiate out beyond the poles into the cytoplasm.
1.1.5 ANIMAL AND PLANT CELLS
Plant and animal cells are structurally very similar because they are both eukaryotic cells.
Similarities : They both contain membrane-bound organelles such as the nucleus, mitochondria, endoplasmic reticulum, golgi apparatus, lysosomes, and peroxisomes. Both are contain similar membranes, cytosol, and cytoskeletal elements.
Differences : Plant cells can be larger than animal cells. the normal range for an animal cell varies from 10 to 30 m while that for a plant cell stretches from 10 to 100 m. Beyond size, the main structural differences between plant and animal cells lie in a few additional structures found in animal cells. These structures include ; chloroplast, the cell wall, and vacuoles.
A detailed description of eukaryotic cells is provided in later chapters.
Friday, August 6, 2010
aithagani srinivas
i MR Aithagani srinivasarao is editor of university express. introduce a new monthly magazine. its useful for every campus.
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