Chapter 3 Microscopy and Cell structure Biology 261 Prof. Santos Fall 2011 • Microscope- tool used to see objects too small to be seen by the naked eye. Types of microscopes used in the study of microorganisms include the light microscope, dark field microscope, phase contrast microscope, confocal microscope, interference microscope, fluorescence microscope, electron microscope, and atomic force microscope. Terms to know • Bright field microscopy- lights rays are used to evenly illuminate the field of view. • Dark field microscopy- light is directed towards the specimen at an angle. This makes it possible for the unstained specimen to appear more visible against a dark background. • Contrast- basically it is the number of visible shades in a specimen. • Microscopes that increase contrast include, phase contrast microscope, interference microscope, dark field microscope, fluorescence, and confocal microscope. Light Microscope 1-Light microscope- uses a beam of light to create an enlarged image of the specimen. The light microscope can either use a mirror or a light bulb to pass light through the specimen. You can magnify a specimen up to 1000x with a good light microscope Parts of the microscope A- body tube- connects the eyepiece with the rotating nosepiece. B-eye piece- the lens the observer looks through. C-rotating nosepiece- holds the objectives and allows you to switch from one to the other. D-objective lens- the lens found on the nosepiece that magnifies the image. Most microscopes contain three; the low power, high power, and the oil immersion lens. • *To determine the total magnification power, multiply the power of the eyepiece with the power of the objective lens. For example; if the eyepiece is 10x and you are using the high power, which is 40x, then the total magnifying power is 400x. So, it means you are looking at the object 400x closer. E-coarse adjustment knob-allows you to move the stage closer to the objective lens and focus the specimen. When you first place a slide in the scope, you should always start with low power and focus with the coarse adjustment. F-fine adjustment knob-allows you to move the stage very slowly and finely focus the specimen under high power. Keep in mind that when you move to high power; the amount of light passing through will diminish. You might need to adjust the diaphragm. G-stage- broad flat platform where you place the slide. H-stage clips- hold the slide in place. I-diaphragm- a circular flat wheel underneath the stage that allows you to control the amount of light passing through. J-arm- used to carry the microscope and for support. K-light source- provides the light needed to create an enlarged image of specimen. The light source is either a mirror or a light bulb. Resolution- the ability of a microscope to separate and show two points that are very close together. Resolution depends on the type of lens used, wavelength of the light, magnification, and specimen preparation. Resolution can be expressed as d= .5 (wavelength)/ NA d= distance between the two points NA = numerical aperture, a mathematical expression that describes how well the condenser directs light rays through the specimen To increase resolution 1- 2- 3- • The maximum resolving power of the best light microscope is .2 um due to the wavelength of light. • When using the 100x objective lens, one must use immersion oil to maximize resolution. This is because of the difference in the refractive index of air and glass. As light rays pass from one medium to another, light rays will bend if there is a difference in the refractive index. • Refractive index is the velocity of light as it travels through a medium. Electron Microscope Electron microscope- uses a beam of electrons to create an enlarged image of the specimen. An electron microscope can magnify a specimen up to 250,000x closer. The wavelength used is less than the wavelength of light, thus the resolution is greater. 2 types • TEM- transmission electron microscope allows one to see fine detail structures in side the cell. • SEM- scanning electron microscope allows one to see the surface details of cells like the membrane proteins. 3-Phase contrast microscope- allows you to see unstained cells by altering the background of the cell. This microscope has a device that allows it to amplifies differences in refractive index to create a contrast. 4-fluorescence microscope Projects ultra violet light causing fluorescent molecules in the specimen to emit a longer wavelength. 5- Confocal microscope Mirrors are used to scan a laser beam across successive regions and planes of a specimen. A computer program constructs a 3D image. 6- Interference microscope causes the specimen to appear as a 3D image. The most common one is the Nomaski differential interference microscope. This microscope has a special device that separates light going through a specimen into 2 beams and then recombines them. The light rays are out of phase when they recombine, yielding a 3D image. 7- Dark field microscope this type of light microscope has a special device that directs light at an angle so that only light scattered by the specimen enters the objective so one sees the specimen against a dark background. 8- Atomic force microscope This very powerful microscope produces a very detailed image of the surface of an specimen by using a very sharp probe (stylus) to move across the surface and “feel” the bumps and valleys of the atoms of the surface. Staining • Use of either a basic or acidic dye to color certain cell parts and make it more visible to the naked eye. Three types of staining techniques; simple, differential and special stains for the flagella, spores, and capsule. Dyes • Basic dyes carry a positive charge. They stain the negative parts of a cell like proteins and nucleic acids. Examples include methylene blue, crystal violet, safranin, malachite green. • Acidic dyes carry a negative charge. They are usually used to stain the background to observe colorless cells. • Neutral no charge • Staining allows you to see parts of the cell you would not ordinarily see. Cells are clear! By adding a stain you are basically coloring certain parts of the cell that take in the dye. Prokaryotic cell Morphology Three basic shapes; spherical called coccus, cylindrical called bacillus and spiral. There are variations! coccobacillus- short rod vibrio- a short curve rod spirochete- a long helical cell with a flexible cell wall and unique mode of utility Pleomorphic- bacteria that vary their shape Grouping Multicellular association 1- fruiting body 2- biofilm Structure of prokaryote cell 1-Flagella provides motility. The flagella is made up of three basic parts, filament, hook and basal body. 2-Pili proteins that enable the bacteruim to adhere to surfaces. Fimbriae allow bacteria to adhere to surfaces and sex pili allow DNA transfer between bacteria. 3-The capsule a viscous and gelatinous layer that surrounds bacteria. It enables bacteria to adhere to certain surfaces and allows organisms to avoid innate defense systems and cause diseases. Ex, Streptococcus pneumoniae. 4- slime layer- gel like layer that is diffuse and irregular. This layer is composed of polysaccharides and enables the bacteria to adhere to surfaces and grow as biofilm. Ex; Streptococcus mutans grows as biofilm on your teeth to form dental plaque. 5- The cell wall a rigid covering consisting of peptidoglycan that gives the bacterium its shape and protection. • The type of cell wall distinguishes between 2 types of bacteria; gram negative and gram positive. • Peptidoglycan is a macromolecule found only in bacteria. • The basic structure of peptidoglycan is an alternating series of 2 major subunits, N- acetylmuramic acid (NAM) and N- acetylglucosamine (NAG). These subunits are covalently bonded to each other to form a glycan chain. • Attached to each NAM molecule is a string of 4 amino acids, a tetrapeptide chain. Cross linkages can form between adjacent chains thus joining adjacent glycan chains. • In gram negative bacteria these tetrapeptides are joined directly. • In gram positive bacteria they are usually joined indirectly by a peptide interbridge. • In gram positive bacteria the peptidoglycan layer is thick. • In gram negative bacteria the peptidoglycan layer is thinner. Teichoic acid • In gram positive bacteria there are polymers of teichoic acid present. These teichoic acid polymers are covalently linked to the NAM molecules of the glycan chain. • These polymers consists of chemically modified ribose or glycerol sugars connected by phosphates. Sugars and D- alanine may be attached to these polymers providing antigenic determination. • Teichoic acid provides rigidity to the cell wall. Outer membrane • In gram negative bacteria there is an outer membrane outside the peptidoglycan. It is a unique lipid bilayer. • The outer membrane is unlike any other membrane. The outside leaflet consists of Lipopolysaccharides instead of phospholipids. • The outer membrane is sometimes called the LPS or lipopolysaccharide layer. • The outer membrane is joined to peptidogylcan by means of lipoproteins • Two parts are importance for medical reasons; a- Lipid A is the portion that anchors the LPS in the lipid bilayer. It plays a role in the immune system. b- O specific polysaccharide is a chain of sugar molecules opposite the Lipid A. Allows for identification. Gram positive periplasm • In gram negative bacteria, it is the space between the inner and outer membrane. Intracellular parts 6- Bacterial chromosome is usually circular double stranded molecule located in a region called the nucleoid 7- Plasmids are small, supercoiled, circular double stranded pieces of DNA that contain few genes. 8- Endospore is a type of dormant cell that resists harsh conditions. 9- Cytoskeleton proteins that provide support. 10- Gas vesicles provide buoyancy 11- granules are accumulations of substances produced in excess. • Examples are glycogen, poly beta hydroxybutyrate, and volutin. • Volutin- a storage form of phosphate. They stain red with methylene blue, sometimes called metachromatic granules. Role is unclear. Thought to be involved in energy storage and pH balance inside the cell. 12- ribosomes involved in protein production Eukaryotic cell 1- Plasma membrane consisting of asymmetrical lipid bilayer. The cell membrane consists of proteins and lipids. Internal protein parts 2- Cilia/flagella are protein structures that consists of microtubules in a 9 +2 arrangement. Cilia and flagella function in motility. 3- cytoskeleton consists of proteins such as microtubules, actin filaments and intermediate filaments that function in cell structure/support and act as a molecular monorail. 4- ribosomes are involved in protein production. 5- Chloroplast- double membrane bound organelle involved in photosynthesis 6- Endoplasmic reticulum is a system of canals involved in the production of macromolecules destined to be secreted to other organelles or outside. Two types, smooth and rough. 7- Golgi Apparatus is a system of flat membranes involved in the modification of material made in the ER. The vesicles are coated with special carbohydrates and phosphate groups that signal the vesicles to their specific location. 9- Nucleus is the control center of the cell. Contains the genetic material and is surrounded by a nuclear envelope. 10- Mitochondrium is the site of cellular respiration 11- Lysosomes 12- peroxisomes are organelles us to oxidize substances, breaking down lipids, and detoxifying certain chemicals.
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