Cells

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[|Eukaryotic Cells]





Cell Measurement
 9700/31O/N12 Q2 b(i).

To calibrate you are trying to give measurable value to the divisions on the eyepiece graticule since this can help you to measure. Remember the eyepiece graticule is viewing images that are already magnified so you have to give them a real value before measuring. Proceed as follows: 1) Place the stage micrometer scale on the microscope stage. (Usually this scale is 1cm long and has either 100 or 50 divisions depending on the type). 2) Super-impose the eyepiece graticule so that you view it in line with the stage micrometer scale underneath (this time the stage micrometer divisions appear much bigger compared to the eyepiece graticule divisions). Count the number of divisions of coincidence between two furthest reference points e.g. 100 eyepiece graticule divisions coincide with 25 stage micrometer @ X40. Considering the fact that the stage micrometer scale has e.g. 1cm (10 mm) representing 100 divisions therefore 10mm = 1000micrometers = 100 divisions. Therefore 1 stage micrometer division = 0.1mm = 100 micrometers. Remember we had 25 stage micrometer divisions coinciding with 100 eyepiece graticule divisions @ X40, this means 25 X 100 micrometers coincide with 100 eyepiece divisions. Therefore 1 eyepiece division = 25 X 100 micrometers /100 eyepiece divisions @ X40. This means 1 eyepiece division @ a magnification of X 40 has a value of 25 micrometers.

However, you need to follow the same procedure @ X100 and X400 magnifications.

Once you know the value of 1 eyepiece graticule division, you can now replace the stage micrometer with the slide then measure the desired area. The number of graticule divisions obtained @ X40 can be multiplied by 25 micrometers (from the calibration).

First you have to match the stage micrometer with the graticule divisions and find how many graticule divisions match one stage division e.g.in Nov.2012 /31 one stage division equals 10 graticule divisions this is why 0.1mm which is the value of one stage division is divided by 10 and therefore 1 graticule division equals 0.01 mm

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Cells
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All living things are made of cells, and cells are the smallest units that can be alive. Life on Earth is classified into five kingdoms, and they each have their own characteristic kind of cell. However the biggest division is between the cells of the **prokaryote** kingdom (the bacteria) and those of the other four kingdoms (animals, plants, fungi and protoctista), which are all **eukaryotic** cells. Prokaryotic cells are smaller and simpler than eukaryotic cells, and do not have a nucleus. Prokaryote = "before carrier bag" i.e. without a nucleus Eukaryote = "good carrier bag" i.e. with a nucleus We'll examine these two kinds of cell in detail, based on structures seen in **electron micrographs** (photos taken with an electron microscope). These show the individual **organelles** inside a cell. __Eukaryotic__ __Cells__

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__Prokaryotic Cells__
 * **Cytoplasm (or Cytosol).** This is the solution within the cell membrane. It contains enzymes for glycolysis (part of respiration) and other metabolic reactions together with sugars, salts, amino acids, nucleotides and everything else needed for the cell to function.
 * **Nucleus**. This is the largest organelle. Surrounded by a __nuclear envelope__, which is a double membrane with __nuclear pores__ - large holes containing proteins that control the exit of substances such as RNA and ribosomes from the nucleus. The interior is called the __nucleoplasm__, which is full of __chromatin__- a DNA/protein complex in a 1:2 ratio containing the genes. During cell division the chromatin becomes condensed into discrete observable __chromosomes__. The __nucleolus__ is a dark region of chromatin, involved in making ribosomes.
 * **Mitochondrion (pl. Mitochondria)**. This is a sausage-shaped organelle (8µm long), and is where aerobic respiration takes place in all eukaryotic cells. Mitochondria are surrounded by a double membrane: the outer membrane is simple and quite permeable, while the inner membrane is highly folded into __cristae__, which give it a large surface area. The space enclosed by the inner membrane is called the mitochondrial __matrix__, and contains small circular strands of DNA. The inner membrane is studded with __stalked particles__, which are the site of ATP synthesis.
 * **Chloroplast**. Bigger and fatter than mitochondria, chloroplasts are where photosynthesis takes place, so are only found in photosynthetic organisms (plants and algae). Like mitochondria they are enclosed by a double membrane, but chloroplasts also have a third membrane called the __thylakoid__ membrane. The thylakoid membrane is folded into __thylakoid disks__, which are then stacked into piles called __grana__. The space between the inner membrane and the thylakoid is called the __stroma__. The thylakoid membrane contains chlorophyll and other photosynthetic pigments arranged in __photosystems__, together with stalked particles, and is the site of photosynthesis and ATP synthesis. Chloroplasts also contain starch grains, ribosomes and circular DNA.
 * **Ribosomes.** These are the smallest and most numerous of the cell organelles, and are the sites of protein synthesis. They are composed of protein and RNA, and are manufactured in the nucleolus of the nucleus. Ribosomes are either found free in the cytoplasm, where they make proteins for the cell's own use, or they are found attached to the rough endoplasmic reticulum, where they make proteins for export from the cell. They are often found in groups called __polysomes__. All eukaryotic ribosomes are of the larger, "80S", type.
 * **Smooth Endoplasmic Reticulum (SER).** Series of membrane channels involved in synthesising and transporting materials, mainly lipids, needed by the cell.
 * **Rough Endoplasmic Reticulum (RER).** Similar to the SER, but studded with numerous ribosomes, which give it its rough appearance. The ribosomes synthesise proteins, which are processed in the RER (e.g. by enzymatically modifying the polypeptide chain, or adding carbohydrates), before being exported from the cell via the Golgi Body.
 * **Golgi Body (or Golgi Apparatus).** Another series of flattened membrane __vesicles__, formed from the endoplasmic reticulum. Its job is to transport proteins from the RER to the cell membrane for export. Parts of the RER containing proteins fuse with one side of the Golgi body membranes, while at the other side small vesicles bud off and move towards the cell membrane, where they fuse, releasing their contents by __exocytosis__.
 * **Vacuoles.** These are membrane-bound sacs containing water or dilute solutions of salts and other solutes. Most cells can have small vacuoles that are formed as required, but plant cells usually have one very large permanent vacuole that fills most of the cell, so that the cytoplasm (and everything else) forms a thin layer round the outside. Plant cell vacuoles are filled with __cell sap__, and are very important in keeping the cell rigid, or turgid. Some unicellular protoctists have __feeding vacuoles__ for digesting food, or __contractile vacuoles__ for expelling water.
 * **Lysosomes.** These are small membrane-bound vesicles formed from the RER containing a cocktail of digestive enzymes. They are used to break down unwanted chemicals, toxins, organelles or even whole cells, so that the materials may be recycled. They can also fuse with a feeding vacuole to digest its contents.
 * **Cytoskeleton.** This is a network of protein fibres extending throughout all eukaryotic cells, used for support, transport and motility. The cytoskeleton is attached to the cell membrane and gives the cell its shape, as well as holding all the organelles in position. There are three types of protein fibres (__microfilaments__, __intermediate filaments__ and __microtubules__), and each has a corresponding __motor protein__ that can move along the fibre carrying a cargo such as organelles, chromosomes or other cytoskeleton fibres. These motor proteins are responsible for such actions as: chromosome movement in mitosis, cytoplasm cleavage in cell division, cytoplasmic streaming in plant cells, cilia and flagella movements, cell crawling and even muscle contraction in animals.
 * **Centriole**. This is a pair of short microtubules involved in cell division. Before each division the centriole replicates itself and the two centrioles move to opposite ends of the cell, where they initiate the spindle that organises and separates the chromosomes (see module 2).
 * **Undulipodium (Cilium and Flagellum)**. This is a long flexible tail present in some cells and used for motility. It is an extension of the cytoplasm, surrounded by the cell membrane, and is full of microtubules and motor proteins so is capable of complex swimming movements. There are two kinds: __flagella__ (no relation of the bacterial flagellum) are longer than the cell, and there are usually only one or two of them, while __cilia__ are identical in structure, but are much smaller and there are usually very many of them.
 * **Microvilli**. These are small finger-like extensions of the cell membrane found in certain cells such as in the epithelial cells of the intestine and kidney, where they increase the surface area for absorption of materials. They are just visible under the light microscope as a __brush border__.
 * **Cell Membrane (or Plasma Membrane).** This is a thin, flexible layer round the outside of all cells made of phospholipids and proteins. It separates the contents of the cell from the outside environment, and controls the entry and exit of materials. The membrane is examined in detail later.
 * **Cell Wall.** This is a thick layer outside the cell membrane used to give a cell strength and rigidity. Cell walls consist of a network of fibres, which give strength but are freely permeable to solutes (unlike membranes). A wickerwork basket is a good analogy. Plant cell walls are made mainly of __cellulose__, but can also contain hemicellulose, pectin, lignin and other polysaccharides. They are built up in three layers called the primary cell wall, the secondary cell wall and the middle lamella. There are often channels through plant cell walls called __plasmodesmata__, which link the cytoplasms of adjacent cells. Fungal cell walls are made of __chitin__ (poly-glucosamine). Animal cells do not have a cell wall, though they do have a layer of carbohydrate outside the cell membrane called the __cell coat__, or __glycocalyx__.

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**Summary of the Differences Between Prokaryotic and Eukaryotic Cells**
 * **Cytoplasm**. Contains all the enzymes needed for all metabolic reactions, since there are no organelles
 * **Ribosomes**. The smaller (70 S) type.
 * **Nucleoid** (or **Nuclear Zone**). The region of the cytoplasm that contains DNA. It is not surrounded by a nuclear membrane.
 * **DNA**. Always circular, and not associated with any proteins to form chromatin.
 * **Plasmid**. Small circles of DNA, used to exchange DNA between bacterial cells, and very useful for genetic engineering.
 * **Cell membrane**. made of phospholipids and proteins, like eukaryotic membranes.
 * **Mesosome**. A tightly-folded region of the cell membrane containing all the membrane-bound proteins required for respiration and photosynthesis. Can also be associated with the nucleoid.
 * **Cell Wall**. Made of __murein__ (not cellulose), which is a glycoprotein (i.e. a protein/carbohydrate complex, also called __peptidoglycan__). There are two kinds of cell wall, which can be distinguished by a __Gram stain__: __Gram positive__ bacteria have a thick cell wall and stain purple, while __Gram negative__ bacteria have a thin cell wall with an outer lipid layer and stain pink.
 * **Capsule** (or **Slime Layer**). A thick polysaccharide layer outside of the cell wall, like the glycocalyx of eukaryotes. Used for sticking cells together, as a food reserve, as protection against desiccation and chemicals, and as protection against phagocytosis.
 * **Flagellum**. A rigid rotating helical-shaped tail used for propulsion. The motor is embedded in the cell membrane and is driven by a H+ gradient across the membrane. Clockwise rotation drives the cell forwards, while anticlockwise rotation causes a chaotic spin. This is the only known example of a rotating motor in nature.
 * ** Prokaryotic Cells ** || ** Eukaryotic cells ** ||
 * small cells (< 5 mm) || larger cells (> 10 mm) ||
 * always unicellular || often multicellular ||
 * no nucleus or any membrane-bound organelles, such as mitochondria || always have nucleus and other membrane-bound organelles ||
 * DNA is circular, without proteins || DNA is linear and associated with proteins to form chromatin ||
 * ribosomes are small (70S) || ribosomes are large (80S) ||
 * no cytoskeleton || always has a cytoskeleton ||
 * motility by rigid rotating flagellum made of flagellin || motility by flexible waving undulipodium, made of tubulin ||
 * cell division is by binary fission || cell division is by mitosis or meiosis ||
 * reproduction is always asexual || reproduction is asexual or sexual ||
 * huge variety of metabolic pathways || common metabolic pathways ||

** Endosymbiosis ** Prokaryotic cells are far older and more diverse than eukaryotic cells. Prokaryotic cells have probably been around for 3.5 billion years, while eukaryotic cells arose only about 1 billion years ago. It is thought that eukaryotic cell organelles like nuclei, mitochondria and chloroplasts are derived from prokaryotic cells that became incorporated inside larger prokaryotic cells. This idea is called **endosymbiosis**, and is supported by these observations: __Cell Fractionation__
 * organelles contain circular DNA, like bacteria cells.
 * contain 70S ribosomes, like bacteria cells.
 * organelles have double membranes, as though a single-membrane cell had been engulfed and surrounded by a larger cell.
 * organelles reproduce by binary fission, like bacteria.
 * organelles are very like some bacteria that are alive today.

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This means separating different parts and organelles of a cell, so that they can be studied in detail. All the processes of cell metabolism (such as respiration or photosynthesis) have been studied in this way. The most common method of fractionating cells is to use **differential centrifugation**:

2. Grind tissue in a blender to break open cells. 3. Filter. This removes insoluble tissue (e.g. fat, connective tissue, plant cell walls, etc). This filtrate is not called a **cell-free extract**, and is capable of carrying out most of the normal cell reactions. ||  ||  ||
 * 1. Cut tissue (e.g. liver, heart, leaf, etc) in ice-cold isotonic buffer. Cold to stop enzyme reactions, isotonic to stop osmosis, and buffer to stop pH changes.

(1 000 x g for 10 min) ||  ||  ||
 * 4. Centrifuge filtrate at low speed

(10 000 x g for 30 min) ||  ||
 * 5. Centrifuge supernatant at medium speed

(100 000 x g for 1 hour) ||
 * 6. Centrifuge supernatant at high speed

(300 000 x g for 3 hours) ||
 * 7. Centrifuge supernatant at very high speed


 * 8. Supernatant is now organelle-free cytoplasm ||

A more sophisticated separation can be performed by **density gradient centrifugation**. In this, the cell-free extract is centrifuged in a dense solution (such as sucrose or caesium chloride). The fractions don't pellet, but instead separate out into layers with the densest fractions near the bottom of the tube. The desired layer can then be pipetted off. This is the technique used in the Meselson-Stahl experiment (module 2) and it is also used to separate the two types of ribosomes. The terms 70S and 80S refer to their positions in a density gradient = **[|Cell & cell organelles]** = [|Target Practice Cell Quiz] =[|Spreadsheet Mitosis]= [|Database ICT]

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