1. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville M I C R O B I O L O G Y WITH DISEASES BY BODY SYSTEM SECOND EDITION Chapter 4 Microscopy, Staining, and Classification

2. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Units of Measurement [INSERT TABLE 4.1]

3. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy General Principles of Microscopy – Wavelength of radiation – Magnification – Resolution – Contrast

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7. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy General Principles of Microscopy – Contrast – Differences in intensity between two objects, or between an object and background – Important in determining resolution – Staining increases contrast – Use of light that is in phase increases contrast

8. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Light Microscopy – Bright-field microscopes – Simple – Contain a single magnifying lens – Similar to magnifying glass – Leeuwenhoek used simple microscope to observe microorganisms

9. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Light Microscopy – Bright-field microscopes – Compound – Use a series of lenses for magnification – Light rays pass through specimen and into objective lens (one of a series of objective lenses) – Oil immersion lens increases resolution because light does not refract – Have one or two ocular lenses – Total magnification = magnification of objective lens X magnification of ocular lens – Most have condenser lens to direct light through specimen

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11. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Light Microscopy – Dark-field microscopes – Best for observing pale objects – Only light rays scattered by specimen enter objective lens – Specimen appears light against dark background – Increases contrast and enables observation of more details

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13. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Light Microscopy – Phase microscopes – Used to examine living organisms or specimens that would be damaged or altered by attaching them to slides or staining them – Treat one set of light rays differently from another set – Light rays in phase produce brighter image, while light rays out of phase produce darker image – Contrast is created because light waves are 1/2 wavelength out of phase

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16. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Light Microscopy – Fluorescent microscopes – Direct UV light source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength – UV light increases resolution and contrast – Some cells and molecules are naturally fluorescent, while others must be stained – Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins

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19. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Light Microscopy – Confocal microscopes – Use fluorescent dyes – Use UV lasers to illuminate fluorescent chemicals in a single plane that is no thicker than 1.0  m – Resolution increased by up to 40% because emitted light passes through pinhole aperture – Computer constructs 3-D image from digitized images

20. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Electron Microscopy – Light microscopes cannot resolve structures closer than 200 nm because shortest wavelength of visible light is 400 nm – Electrons have wavelengths of 0.01 nm to 0.001 nm, so electron microscopes have greater resolving power and greater magnification – Magnifies objects 10,000X to 100,000X – Gives detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms – Two types – Transmission electron microscopes – Scanning electron microscopes

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24. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy Probe Microscopy – Uses minuscule, pointed, electronic probes to magnify more than 100,000,000 times – Two types – Scanning tunneling microscopes – Atomic force microscopes

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26. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Microscopy [INSERT TABLE 4.2]

27. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Staining Increases contrast and resolution by coloring specimens with stains/dyes Smear of microorganisms (thin film) air dried to slide and then fixed to surface by heat or chemical fixation Microbiological stains usually salts composed of cation and anion, with one colored (chromophore) Acidic dyes stain alkaline structures; more commonly, basic dyes stain acidic structures

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29. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Staining Simple stains Differential stains – Gram stain – Acid-fast stain – Endospore stain Special stains – Negative (capsule) stain – Flagellar stain

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37. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Staining Staining for Electron Microscopy – Chemicals containing heavy metals used for transmission electron microscopy – Stains may bind molecules in specimens or the background

38. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Classification & Identification of Microorganisms Taxonomy consists of classification, nomenclature, and identification Enables scientists to organize large amounts of information about organisms and make predictions based on knowledge of similar organisms

39. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Classification & Identification of Microorganisms Linnaeus, Whittaker, and Taxonomic Categories – Linnaeus – Provided system that standardized the naming and classification of organisms based on characteristics in common – Grouped similar organisms that can successfully interbreed into categories called species – Used binomial nomenclature in his system

40. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Classification & Identification of Microorganisms

41. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Classification & Identification of Microorganisms Linnaeus, Whittaker, and Taxonomic Categories – Whittaker – Linnaeus proposed only two kingdoms – Whittaker proposed a widely accepted taxonomic approach based on five kingdoms: Animalia, Plantae, Fungi, Protista, and Prokaryotae

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43. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Classification & Identification of Microorganisms Linnaeus, Whittaker, and Taxonomic Categories – Linnaeus’s goal was classifying and naming organisms as a means of cataloging them – More modern goal is understanding relationships among groups of organisms – Major goal of modern taxonomy is to reflect phylogenetic hierarchy – Greater emphasis on comparisons of organisms’ genetic material led to proposal to add domain

44. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Classification & Identification of Microorganisms Domains – Carl Woese compared nucleotide sequences of rRNA subunits (changes occur rarely) – Proposal of three domains based on three basic types of cells as determined by ribosomal nucleotide sequences – Three Domains: Eukarya, Bacteria, and Archaea – Cells in the three domains also differ with respect to many other characteristics

45. Copyright © 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings Classification & Identification of Microorganisms Taxonomic and Identifying Characteristics – Physical characteristics – Biochemical tests – Serological tests – Phage typing – Analysis of nucleic acids

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