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Techniques & Tools I. Types of Microscopes 1. Light Microscopes A. Simple – B. B. Complex – C. Visual Light. 3 objectives, low, high, oil immersion. Magnification – Resolving Power Light Microscopes Review Why is a light microscope called a light microscope? Name the two lenses in a compound microscope. 2. U.V. light for illumination Magnification Resolving . . 3. Fluorescent light with specimens that’s been stained with Magnification Resolving - 4. Dark-field of visible light, light is refracted by specimen as light enters the objective. Mag Resolving - . . 5. Phase Contrast Microscope Observe matter, unstained. Detects changes (phases) in visible light as it through a specimen. Mag Resolving - 6. Transmission Electron Microscope (TEM) Beam of Thin specimen that’s been stained with electrons opaques heavy metal. Travels specimen. Viewed on screen. Mag - Resolving - 7. Scanning Electron Microscope (SEM) Beam of . surface & produces a image of specimen. Mag - Resolving Scanning Tunneling Electron Micrograph Review What is the difference between magnification and resolution? Which microscope gives the greatest magnification? What is the magnification limit most light microscopes? II. Techniques for Microscopic Staining 1. Living Microscopic Organisms. A. Some Bacteria are mobile, others are not but appear to move. 1. movement due to air or water molecules hitting the microorganism. B. Non Staining Techniques - hard to see. Ex. 2. Stained Microorganisms Colored by a chemical dye to make visible. General Techniques for all staining. 1. Smear - slide out the drop with the bacteria in it. 2. Stained - reveal size, shape, arrangement & presence of some internal structures. Types of Staining 1. Simple A. Smear. B. Single stain is used (Sudan Black). 2. Differential Staining A. Gram 1. 2. 3. 4. . 5. . - decolorizes after alcohol wash, need a counter stain (red, safranin). stain. - retains blue B. Acid Fast - Staining Used for the genus . Retain carbolfuchsin (red)when washed with acidic alcohol. Counter with . Gram Staining Review What Why is a differential stain? is a Gram stain so useful? What structure of the cell determines its Gram staining properties? III. Bacterial Morphology Shapes are recognized, but others exist. 1. - round A. pairs B. chains C. cubes • Depending when they divide and then adhere to each other. 2. - rod or cylinder shape. 3. Spiral Forms twisted rods or cylinders A. - actual spirals (corkscrew); rigid. B. - flexible. Acid Fast Stain Carbol Fuchsin - 5minutes Wash with tap water. Acid – 2 minutes. Wash with tap water. Methylene Blue – 2 minutes. Wash with tap water. Blot dry. Acid Fast bacteria do no decolorize with acid alcohol. They appear red against a blue background. 4. - Extensions on their surface which gives them a star appearance. With age bacteria morphology may change - swell or show rudimentary branching. IV. Reproduction ( reproduction) - one cell divides into two identical daughter cells. Each cells lives on their own after replication even though they may not be Prokaryotes Surface Layers - Capsule Review How do bacteria mainly reproduce? What is the advantage of that type of reproduction? What is the disadvantage of that type of reproduction? 2. Cell Wall •Function is to hold the cell together. Composed of sub-units found nowhere else in nature. Produces symptoms of diseases. Site of action of some of the most effective antibiotics. - Common structural component made up of sugar & amino acids that makes the cell wall rigid. Components, two different sugars. A. B. Differences in staining properties. determines the gram- – effective against cell walls of bacteria. V. Bacterial Structure A. - Collectively known as the 1. - material secreted by bacteria that adheres to the exterior of the bacterial cell. Functions Antigenic Possible Waste products A. - organized, thickened material around each cell or pairs of cells. Ex. - Streptococcus pneumoniae - can not survive in a host unless it can synthesize a capsule, because the host will quickly destroys it. B. - Unorganized loosely attached polysaccharide. C. - oral bacteria that secrete glycocalyx. Review What is the function of the cell wall? What are the affects of penicillin on bacterial cell walls? What are the cell walls made of? Where is the only place on earth that this component is found at? A. Gram Positive ( ) 1.Numerous layers of peptidoglycan (up to ). 2. associated in the peptidoglycan layer. B. Gram Negative ( ) 1. Outer layer consists of a that consists of several other molecules. 2. Lipopolysaccharides (LPS), ,& Porin proteins. 3. Cell Membrane or Plasma membrane. Located inside the cell wall. Composition is 60% protein & 40% Lipids. Functions 1. Osmotic regulator. 2. Enzymes necessary for the synthesis & transport of peptidoglycan, teichoic acid & other membrane components. 3. Secretes extracellular hydrolytic enzymes. 4. Ensures segregation of nuclear material during cell division. 5. Transport of electrons & protons that are released during aerobic oxidation & turns it into chemical energy that can be used by the cell. 6. Barrier to the entry of most molecules into the cell (nutrients entering & wastes leaving). A. Semi-permeable allows some things to pass, but not others. 1. Diffusion - do not use energy. a. Passive or Simple Diffusion -molecules flow freely in & out of the cell (Small). b. Facilitated Diffusion - Use membrane transport proteins (Permeases) for molecules to move in & out of the cell (Large). 2. Active transport - uses energy to cross the membrane. Several proteins are required for this. Surface binding proteins. Membrane transport proteins. B. -structures that extend from the surface. 1. - long slender , protein used for locomotion. Bacteria can move lengths /minute (6ft man running 82 mph). A. Composed of three parts. 1. 2. 3. B. Types of flagella arrangements. C. movement toward food substances or away from harmful substances. Toward- straight line Away - tumble away - Due to the of the flagella movement ( ) Possible proteins on the cytoplasm D. - responds to various amount of light. E. - responds to various amount of oxygen. F. - reacts to the earth’s magnetic field due to a row of magnetic particles. Line up north-south direction. Chemotaxsis 2. - shorter & thinner than flagella. Functions 1. Movement of reproduction. 2. during to surfaces. sexual C. Cytoplasm 1.Cytoplasm water, other 20% Nucleic Acids, Proteins, Carbohydrates, Lipids. Primary site of synthetic synthesis of proteins 2. - singular circular double strand of DNA that is supercoiled. that the Bacteria. , but histonelike structures. No or useless DNA. 3. - Small circular pieces of double stranded DNA that contain genetic information to resistance of particular antibiotics. 4. - Site of protein synthesis Composed of 60% RNA & 40% Protein. 5. 6. - Not integral parts of the cell structure A. - reserve source of phosphate & energy (Volutin). B. Granulose C. Others - Sulfur, lipids, glycose. - special membranes systems found in certain photosynthetic bacteria & cyanobacteria that contain pigment concerned with photosynthesis. D. Special Structures 1. Minute highly durable body formed with in the cell & capable of development into a new vegetative organism. adverse conditions Outer layers Cortex layers to Lack many enzymes; but may have some in small amounts. amounts of ,high calcium, protein & Polysaccharide antigens. Endospore Formation Capsule & Endospore Staining Directions CAPSULE Crystal Violet 2-5 minutes Drain extra crystal violet in sink. Wash with copper sulfate for 30 seconds. Dry. Endospore Stain with toluidine blue for 5-15 minutes. Rinse with tap water. Dry. VI. Preparation of a Pure Culture Culture is a medium in which microorganisms can grow on. Pure Cultural Techniques 2 Techniques are commonly used. 1. 2. -General preparation. . Inoculating a dilution of the mixed cultures in to a melted agar. Poured onto a sterile petri dish. Bacteria grow sedately (isolated) in a solid medium. Help ensure pure culture. VII. Types of Culture Media pH and temperature must be controlled in all media 1. 2. – Common Boiling ground meat with water and filtering off the solid material-leaves a clear liquid infusion. Make solid by adding agar to it. Artificial or Complex Medium - beef extracts, yeast, blood Synthetic or Defined Medium - can write the chemical formula for. broth. - Various sugars are added to the nutrient 3. Selective & Differential Media A. - Chemicals are added to the medium to inhibit the growth of some bacteria while letting others grow. B. - Acid indicator is added so that many colonies that formed acid will turn colors. 4. (Liquid) - Help the growth of certain bacteria and not others. VII. Oxygen Requirements 1. 2. 3. 4. grow. - will not grow in the presence of free oxygen, may even be killed. oxygen. -prefers the presence of low lives in the presence of both. a. b. 5. - require free oxygen to - produce lactic acids. - carbon dioxide. - will grow in the presence of oxygen but do not posse an oxidative metabolism. - VIII. Sterilization Methods No living organisms are in the media when inoculated. 1. Autoclave-steam under pressure 15lbs/in2. 2. Filter.