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Microscopy. Procaryote anatomy: membranes & internal structures.

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Last revised: Friday, January 7, 2000
Reading: Ch. 2; Ch. 3 (p. 37-51) in Prescott et al, Microbiology, 4th Ed.
Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!
Copyright 2000. Thomas M. Terry

Microscopy

Units of Measurement

Size and Scale

Resolving Power

Optical Instrument Resolving Power RP in Angstroms
Human eye 0.2 millimeters (mm) 2,000,000 A
Light microscope 0.20 micrometers (Ám) 2000 A
Scanning electron microscope (SEM) 5-10 nanometers (nm) 50-100 A
Transmission electron microscope (TEM) 0.5 nanometers (nm) 5 A

Light Microscopes

  1. Bright-Field Microscope
    gramstain image
    • Advantages: convenient, relatively inexpensive, available
    • Disadvantages: resolving power 0.2 micrometers at best, can recognize cells but not fine details
    • Needs contrast; cells are mainly water. Easiest way to view cells is to fix and stain.
    • Fixation
      • preserves cells; disrupts proteins, prevents decay/degradation.
      • typical treatments: heat, formalin, glutaraldehyde
    • Staining
      1. Simple Stains
        • adds colored compounds --> contrast
        • basic dyes: e.g. methylene blue, crystal violet. Cations (have + charges)----bind to - charge groups on proteins, nucleic acids
        • acidic dyes: e.g. eosin, acid fuchsin. Anions (have - charges); bind to + charges on proteins, phospholipids
      2. Differential Stains
        • allow differentiation between different organsisms. Examples:
        • Gram stain----lab this week. Distinguishes 2 major groups of bacteria: + and -.
        • Spore stain----lab next week. Malachite green binds specifically to compounds in endospore wall.
        • Acid fast stain----not done in 229 lab. Allows detection of some bacteria with waxy coat (Mycobacteria).
    • Optical path: source, condenser lens, specimen, objective lens, eyepiece lens

  2. Phase-Contrast Microscope
    gramstain image
    • Cells are mostly water, very little contrast from surrounding medium, so not very visible in light
    • Phase scope converts slight diffs. in refractive index and cell density into variations.
    • Scope uses annular stop below condenser: thin transparent ring in opaque disk --> hollow light cone. As light passes through specimen, some rays are deviated and retarded by 1/4 wavelength.
    • Have phase plate in objective lens: transparent optical disk with phase ring.
    • Undeviated light passes through ring, is advanced by 1/4 wavelength --> bright background. But deviated light doesn't pass through phase ring, so is not advanced. When light gets focused, deviated rays cancel out with undeviated rays, producing dark image where objects were.
    • Advantage: can see live material w/o staining
  3. Fluorescence Microscope
    gramstain image
    • Fluors are chemicals that adsorb light to produce excited electrons, later reradiate light = flourescence.
    • To use, need special type of microscope. Must illuminate with ultraviolet or violet light (--> excited fluor). Need filters to remove this light from light traveling to ocular lens; only fluoresced light emitted from object will then appear to eye. Need dark field condenser to create dark background.
    • Can couple flour to specific probe molecules (usually antibodies), bind to preparation. If sample is illuminated with wavelength of exciting light, then filter out that wavelength to prevent reaching sample, see nothing. But if fluorescence occurs, diff. wavelength light produced, see object.
    • good technique to detect specific microbe in complex sample. (e.g. detect gonococcus in vaginal smear). Requires correct microscope, fluors, technical skill.
  4. For further information, see "Cell structure and Microscopy"

Electron Microscope

Transmission Electron Microscope (TEM)

Specimen Preparation

Scanning Electron Microscope (SEM)

Specimen Preparation


Prokaryote Anatomy

Composition of a bacterial cell

How big are bacteria?


Basic Structures of Prokaryotic Cells.

  1. Overview: view model of the bacterial cell

Cell membrane

Membrane Components and Structure

  1. Chemical Composition of membranes: ~50% lipid, 50% protein
    • Membrane lipids are typically phospholipids, contain two fatty acids joined to glycerol by ester bonds (See Fig. 2.7 in text).
    • View interactive pdb file of phospholipid. (Note: to view this file on your computer, the Chime plug-in must be installed in your plug-in folder.)
    • Phospholipids assemble into a lipid bilayer.
    • Biological membranes contain lipid bilayer + many embedded proteins. Proteins "float" in fluid lipid layer = "fluid mosaic model" of membrane structure.
    • Other membrane lipics: sterols and hopanoids, rigid planar molecules, may add strength to membranes. (See Fig. 3.6) Sterols usually absent in bacterial membranes (common in eukaryotic membranes, account for 5-20% of total lipid). But hopanoids present, probably stabilize membranes. Enormous % of fossil fuels = hopanoids (global mass est. at 1011-12 tons, about same as total mass of organic carbon in all living organisms, ~ 1012 tons!)
    • Note: Archaea have different type of lipids; not fatty acids, instead built of isoprene units. (See text Fig. 3.19). Typical membrane lipids are called diglycerol diethers or glycerol tetraethers (see Fig. 20.3). The latter molecule produces lipid monolayers instead of bilayers.
    • View physiology of Archaea. (Note: you need a password to the Biology Place to visit this site.)
    • Membrane proteins contribute stability and many functions, such as transport.

Inside the Cell

Cytoplasm

Nucleoid

Inclusion bodies (found in some, not all, cells)

  1. stored energy
    • poly-beta-hydroxybutyric acid (fatty material, very high energy)
    • glycogen (polysaccharide, made of glucose sugare; good energy source)
    • polyphosphate (used to store phosphate, often a limiting nutrient)
    • sulfur granules (common in some photosynthetic bacteria)
    • magnetosomes (common in magnetotactic bacteria); allow cells to orient to magnetic fields
  2. gas vesicles
    • found in many photosynthetic bacteria and cyanobacteria
    • involved in flotation: form rigid air-filled sac
    • surrounded by rigid protein (not lipid) membrane. Impermeable to liquids, but permeable to gas.

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