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Chapter 2

Chapter 2. Observing the Microbial Cell. 10 m m. Observing Microbes. Human eyes have limited resolution Resolution = 150 m m (1/7 mm, 1/200 inch) Microscope needed to see smaller objects Eukaryotic microbes Protozoa, algae, fungi 10–100 m m Prokaryotes Bacteria, Archaea

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Chapter 2

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  1. Chapter 2 Observing the Microbial Cell

  2. 10 mm Observing Microbes • Human eyes have limited resolution • Resolution = • 150 mm (1/7 mm, 1/200 inch) • Microscope needed to see smaller objects • Eukaryotic microbes • Protozoa, algae, fungi • 10–100 mm • Prokaryotes • Bacteria, Archaea • 0.4–10 mm

  3. Relative sizes of different cells

  4. Bacterial cell morphologies • cocci • rods, bacilli • spirilla • vibrios • spirochetes • irregula shapes

  5. Optics and Properties of Light • Visible light has wavelengths 400–750 nm • Maximum resolution is 1 wavelength • Magnification spreads light rays out • To 150 mm, resolution of our eyes • Distance between photoreceptor cells

  6. Optics and Properties of Light • Refraction • Passage through lens material bends light • Parabolic lens brings rays to a focus point • Lens with 2 differently shaped convex sides magnifies image

  7. Bright-Field Microscopy • Increasing resolution • Use shorter wavelength light • UV, X-rays • But images aren’t visible to human eye • Optimize contrast • Differentiate between objects • Lens quality • Collects more light from specimen • Wider lens closer to specimen • Higher numerical aperture (NA) • Use immersion oil NA = n sin

  8. Bright-Field Microscopy • Increasing resolution • Multiple lenses • Correct each other’s aberrations • Compound microscope • Need to focus two lenses • Objective • Condenser

  9. Staining • Fix cells to hold in position • Stain with dye • Reacts with chemical structure of organism • Gram stain reacts with thick cell wall • Increases absorbance • Easier to find in low-contrast conditions Gram-negative cells Gram-positive cells

  10. Dark-Field Microscopy • Light shines at oblique angle • Only light scattered by sample reaches objective • With enough light, some bounces off object • Even objects smaller than wavelength of light • Makes visible objects below resolution limit • Flagella, very thin bacteria Helical bundle of flagella

  11. Phase-Contrast Microscopy • Light passes through and around sample • Light through sample is refracted • Changes phase of light • Light waves out of phase cancel • Sample appears dark against light background • Shows internal organelles of eukaryotes

  12. Differential Interference Contrast (DIC) Microscopy • Polarized light passes through specimen • Sample boundaries bend light • Second polarized lens blocks light • Bent light affects brighter or darker than background Cell nuclei Head of microscopic worm (C. elegans) Bacterium Pharynx (mouth) 10 mm

  13. Fluorescence Microscopy • Fluorophores absorb high-energy light • Emit lower-energy light • Label molecules of interest in cell • Marker for position of molecules within cell UV blue green blue green red

  14. Fluorescently Labeling Molecules • Attach directly to some molecules • DAPI binds DNA • Attach labeled antibody to molecules • Antibody binds specific molecules • Fluor covalently bound to antibody

  15. Fluorescently Labeling Molecules • Attach labeled nucleotides to DNA • Nucleotide probe base-pairs to DNA • Fluor covalently bound to probe • Gene fusion • Protein of interest fused to fluorescent protein • GFP from jellyfish

  16. Electron Microscopy • Electrons behave like light waves • Very high frequency • Allows very great resolution • A few nanometers • Sample must absorb electrons • Coated with heavy metal • Electron beam and sample are in a vacuum • Lenses are magnetic fields

  17. Transmission EM • Sample is fixed to prevent protein movement • Aldehydes to fix proteins • Flash-freezing • High-intensity microwaves • Fixed sample is sliced very thin • Microtome • Sample is stained with metal • Uranium • Osmium

  18. Transmission EM • High resolution • Can detect molecular complexes • Ribosomes • Flagellar base • Strands of DNA • Need many slices to determine 3D structure

  19. Scanning EM • Sample is coated with heavy metal • Not sliced • Retains 3D structure • Gives 3D image • Only examines surface of sample

  20. Atomic Force Microscopy

  21. Visualizing Molecules • X-ray crystallography • Locates all atoms in a large molecular complex • Sample must be crystallized • Nuclear magnetic resonance (NMR) • Measures resonance between chemical bonds • Can locate all atoms in a small protein • Shows atomic movement of proteins in solution • Proteins embedded in membranes

  22. X-Ray Crystallography • X-rays have tiny wavelength • Resolution less than 1 Angstrom • No lenses to focus X-rays • Shoot X-rays at crystallized sample • Many molecules in identical conformation • X-rays diffract according to position of atoms • Compute position of atoms from pattern of scattered X-rays

  23. X-Ray Crystallography • Can detect position of thousands of atoms in a complex of proteins “Ribbon” shows position of “backbone” of amino acids in proteins

  24. Ranges of resolution

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