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UMass Amherst MICROBIO 310 - Modern Microbiology and Ways to View Microbes

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MICROBIO 310 1st Edition Lecture 4Outline of Last Lecture I. 1.5 The Impact of Microorganisms on HumansII. 1.6 The Historical Roots of MicrobiologyIII. 1.7 Pasteur and the Defeat of Spontaneous Generation IV. 1.8 Koch, Infectious Disease, and Pure Culture Microbiology Outline of Current Lecture I. 1.9 The Rise of Microbial DiversityII. 1.10 The Modern Era of MicrobiologyIII. 2.1 Some Principles of Light MicroscopyIV. 2.2 Improving Contrast in Light MicroscopyV. 2.3 Imaging Cells in Three DimensionsVI. 2.4 Electron MicroscopyCurrent Lecture1.9 The Rise of Microbial Diversity• Sergei Winogradsky (1856–1953) and the Concept of Chemolithotrophy– Demonstrated that specific bacteria are linked to specific biogeochemical transformations (e.g., S & N cycles)– Proposed concept of chemolithotrophyenergy from inorganic compounds instead of the sun• Oxidation of inorganic compounds linked to energy conservationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.1.10 The Modern Era of Microbiology • Major Subdisciplines of Applied Microbiology:– Medical microbiology and immunology • Have roots in Koch’s work– Agricultural microbiology and industrial microbiology• Developed from concepts developed by Beijerinck and Winogradsky– Aquatic microbiology and marine microbiology • Developed from advances in soil microbiology– Microbial ecology• Emerged in 1960s–70s • Basic Science Subdisciplines in Microbiology:– Microbial systematics• The science of grouping and classifyingmicroorganisms– Microbial physiology• Study of the nutrients that microbes require for metabolism and growth and the products that they generate– Cytology• Study of cellular structure– Microbial biochemistry• Study of microbial enzymes and chemicalreactions– Bacterial genetics• Study of heredity and variation in bacteria– Virology• Study of viruses• MolecularMicrobiology– Biotechnology• Manipulation of cellular genomes• DNA from one organism can be inserted into a bacterium and the proteins encoded by that DNA harvested– Genomics: study of all of the genetic material (DNA) in living cells (switches in genomeare important)• Transcriptomics: study of RNA patterns• Proteomics: study of all the proteins produced bycell(s)• Metabolomics: study of metabolic expression in cells2.1 Some Principles of Light Microscopy - Compound light microscope uses visible light to illuminate cells• Many different types of light microscopy:– Bright-field– Phase-contrast – Dark-field– Fluorescence- Bright-field scope – Specimens are visualized because of differences in contrast (density) between specimen and surroundings • Two sets of lenses form the image – Objective lens and ocular lens– Total magnification = objective magnification X ocular magnification– Maximum magnification is ~2,000x• Resolution: the ability to distinguish two adjacent objects as separate and distinct– Resolution is determined by the wavelength of light used and numerical aperture of lens– Limit of resolution for light microscope is about 0.2 um2.2 Improving Contrast in Light Microscopy• Improving contrast results in a better final image• Staining improves contrast– Dyes are organic compounds that bind to specific cellular materials– Examples of common stains are methylene blue, safranin, and crystal violet- Staining a slideI. Preparing a smear– Spread culture in thin film over slide and dry in airII. Heat fixing and staining– Pass slide through flame to heat fix– Flood slide with stain; rinse and dryIII. Microscopy– Place drop of oil on slide and examine it with 100x objective lens• Differential stains: the Gram stain• Differential stains separate bacteria into groups• The Gram stain is widely used in microbiology – Bacteria can be divided into two major groups: o gram-positive (G+) o gram-negative (G-)– Gram-positive bacteria appear purple after staining (gram-positive is thick because it has a lot of peptidoglycan-cell wall component)– Gram-negative bacteria appear red after staining • Phase-Contrast Microscopy– Phase ring amplifies differences in the refractive index of cell and surroundings– Improves the contrast of a sample without the use of a stain– Allows for the visualization of live samples– Resulting image is dark cells on a light background• Dark-Field Microscopy– Light reaches the specimen from the sides– Light reaching the lens has been scattered by specimen– Image appears light on a dark background– Excellent for observing motility• Fluorescence Microscopy– Used to visualize specimens that fluoresce• Emit light of one color when illuminated with another color of light – Cells fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye like DAPI– Widely used in microbial ecology for enumerating bacteria in natural samples2.3 Imaging Cells is Three Dimensions• Atomic Force Microscopy (AFM)– A tiny stylus (needle) is placed close to a specimen– The stylus measures weak repulsive forces between it and the specimen– A computer generates an image based on the data received from the stylus2.4 Electron Microscopy• Electron microscopes use electrons instead of photons to image cells and structures– Transmission electron microscopes (TEM) – Scanning electron microscopes (SEM)• Transmission Electron Microscopy (TEM)– Electromagnets function as lenses– System operates in a vacuum– High magnification and resolution (0.2 nm)– Enables visualization of structures at the molecular level– Specimen must be very thin (20–60 nm) and be stained• Scanning Electron Microscopy (SEM)– Specimen is coated with a thin film of heavy metal (e.g., gold)– An electron beam scans the object– Scattered electrons are collected by a detector and an image is produced– Even very large specimens can be observed– Magnification range of


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