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BIOL 2905 (101)
Chapter 4

Chapter 4 Microscopy.docx

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BIOL 2905
Tanya Da Sylva

Chapter 4 Microscopy  Identify the two primary metric units used to measure the diameters of microbes - One tenth of a centimeter is millimeter(mm) - So one-thousandth of a millimeter is a micrometer - One thousand of a micrometer is a nanometer ** Discuss the relationship between wavelength and resolution - Beams of radiation may be referred to as either rays or waves. These various forms of radiation differ in wavelength,- the distance between to corresponding parts of the wave - Ex the human eye discriminates among different wave lengths of visible light and sends patterns of nerve impulses to the brain ex we see violet at 400nm and red at 650 - Moving electrons act as wave lengths - Resolution also called resolving power is the ability to distinguish objects that are close together ex Optomitrist eye chart has a resolution of 20 feet, Leeuwenhoks microscope had resolving power of 1 um meaning if they were closer together than one um they would look like they are together. - Resolution distance is dependent on - 1) the wavelength of the electromagnetic radiation - 2) numerical aperture (ability of the lens to gather light) - Resolution distance = 0.61/numerical aperture iscuss the relationship between contrast and staining in microscopy Contrast –difference in intensity between two objects or between an object and its background - Important in determining resolution for example if two golf balls are lying in grass easier ti see from a distance than if it was on a white towel - Microorganisms are colorless and have little contrast whether light or electrons are used so staining is most often used - It would be much easier if these balls were painted black be able to distinguish from each other and their backround for microscopic viewing - Smear- smear you want see what you have so one a slide you put a drop of the stuff, air dried then viewed - Heat fixation- slide is gently heated by passing the slide under a Bunsen burner - Chemical Fixation involves applying a chemical such as methyl alcohol to the smear for a min, the fixation and drying will kill the organism and able to stick to the slide- preserve shape and size. - **When using electron microscopy- dried cause water vapors can stop electron beam. Dyes used in light microscopy usually use salts so an anion and cation - At least one of the ions in the molecular makeup of dyes is colored - This colored portion is called Chromophore - Chromophore bind to chemicals via covalent, ionic, or hydrogen bond - Ex Methylene blue chloride is composed of catonic chromophore, Methylene blue and a chloride anion called basic dyes combine with the stain acidic structure work best under basic conditions - Because Methylene blue is pos charged, it ionicalllly bonds too neg charged molecules in cells including DNA and many proteins - Aionic dyes, eosin bind to positively charged molecules such as amino acid also called acidic dyes cause they stain alkaline structures and work best in acidic environment Describe simple, Gram, acid-fast and endospore staining procedures - Simple- single basic dye ex-crystal, violet, safranin or Methylene blue - Soak in dye for 30-60s, pat dry, view for size shape and arrangement of shape - Gram - - 1) 1min rinse with water,1min iodine is a mordant makes it less soluble, 10-30sec rinse- acts as a decolorizing agent, breaks down wall of gram- allowing stain and mordant to be washed away easily Acid-Fast Stain cells of the genera Mycobacterium and Nocardia cause most of human disease including t.b, leprosy and other lung and skin infection - Have large amounts of waxy lipids in their cells walls so can’t really satin with Gram - Cover smear with small piece of tissue paper to retain dye during procedure - Step 1 – heat used to drive stain through the waxy wall and into the cell- remaining trapped - Step 2 add a solution of Hydrochloric acid ph 1.0 and alcohol –removes color from non-acid-fast cells and the background acid fast cells remain red acid can’t go past waxy wall - Stains only bleached- non acid fast cells - Staining procedure results in pink acid-fast - Ex presence of acid-fast bacilli (AFBs) in sputum – mycobacterial infection - Endospore Stain A special stain technique is used to examine bacterial spores. Malachite green is used with heat to force the stain into the cells and give them color. A counter stain, safranin, is then used to give color to the non spore forming bacteria. At the end of the procedure, spores stain green and other cells stain red. Flagellular stain For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Flagella so stained can then be observed. -Purpose of classifying organisms- bring sense of order and organization to the a variety and diversity of living things, enhance communication to make prediction about the structure and function of similar organisms and to understand and uncover certain evolutionary connections - Sort organisims on the basis of mutual similarities into overlapping groups called taxa - Taxonomy consist of classification, assigned of organisms to taxa – based on similarities, nomenclature=- practical science of determining that an isolated individual or pop belongs to a particular taxon uss the difficulties in defining species of microorganisms Its hard to define species because as it states species means “A group of organisms that interbreed to produce viable offspring” this would work for more complex animals but not satisfactory for asexual organisms- meaning most microbes - Some scientist classify them as strains ( a pop of cells that arose from a single cell that share stable properties, differ from other strains and evolve as a group from general to specific Eukarya, Bacteria, and Archea - All have different rRNA sequences - - differ including lipids in cell membrane, transfer RNA(tRNA) molecules, and sensitivity to antibiotics cedures taxonomists use to identify and classify microorganisms 1. MORPHOLOGY---This refers to the form of the organism---in bacteria this is the basic shape, the size, any characteristic arrangement, the presence of flagella, presence of a capsule, presence of endospores, etc. This is a beginning in classifying and identifying microbes, but it is of limited use. Many microbes look exactly the same. Even eubacteria and archaea may look identical through the microscope. Based on morphology alone, it is sometimes even quite difficult to determine which group of microbes an organism belongs to. An example of this is an opportunistic pathogen found mainly in AIDS patients, Pneumocystis jiroveci, previously known as Pneumocystis carinii. At first, this organism was believed to be a protozoan, but more recently it has been found to be more closely related to fungi. This is important because it may allow more effective treatment. 2. DIFFERENTIAL STAINING---this typically begins with a gram stain. The acid-fast stain is another example. 3. BIOCHEMICAL TESTS---since so many bacteria look so much alike, it is frequently necessary to resort to biochemical tests for identification. What these tests are really looking for is the ability of an organism to produce certain enzymes. They do this by testing for the ability of a species to make use of certain nutrients, and also what was
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