Microbial Cell Structure Function

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Microbial Cell Structure Function Notes

Transcript of Microbial Cell Structure Function

  • Microbial Cell Structure and Function

    01-20-15

  • I. Microscopy

    2.1 Discovering Cell Structure: Light Microscopy

    2.2 Improving Contrast in Light Microscopy

    2.3 Imaging Cells in Three Dimensions

    2.4 Probing Cell Structure: Electron Microscopy

  • Microbial Size

    http://learn.genetics.utah.edu/content/begin/cells/scale/

  • Microscopy for Different Size Scales

    Different microscopes are required to resolve various cells and subcellular structures

  • 2.1 Discovering Cell Structure: Light Microscopy

    Compound light microscope uses visible light to illuminate cells

    Many different types of light microscopy:

    Bright-field

    Phase-contrast

    Dark-field

    Fluorescence

    Animation: Light Microscopy

  • 2.1 Discovering Cell Structure: Light Microscopy

    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 ocular magnification

    Maximum magnification is ~2,000

    Ocularlenses

    Objective lens

    Stage

    Condenser

    Focusing knobs

    Light source

    Specimen onglass slide

  • Visualized

    image

    Eye

    Ocular lens

    Intermediate image

    (inverted from that

    of the specimen)

    Objective lens

    Specimen

    Condenser lens

    Light source

    None

    100, 400,

    1000

    10

    10, 40, or

    100 (oil)

    Magnification Light path

  • 2.1 Discovering Cell Structure: Light Microscopy

    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 m

  • 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

    Animation: Staining

    Animation: Microscopy & Staining Overview

  • I. Preparing a smear

    II. Heat fixing and staining

    III. Microscopy

    Spread culture in thin

    film over slide

    Pass slide through

    flame to heat fix

    Dry in air

    Flood slide with stain;

    rinse and dry

    Place drop of oil on slide;

    examine with 100

    objective lens

    Slide Oil

  • 2.2 Improving Contrast in Light Microscopy

    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: Gram-positive and Gram-negative

    Gram-positive bacteria appear purple and Gram-negative bacteria appear red after staining

  • Step 1

    Result:

    All cells purple

    Result:

    All cells

    remain purple

    Result:

    Gram-positive

    cells are purple;

    gram-negative

    cells are colorless

    Result:

    Gram-positive

    (G+) cells are purple;

    gram-negative (G-) cells

    are pink to red

    Flood the heat-fixed

    smear with crystal

    violet for 1 min

    Add iodine solution

    for 1 min

    Decolorize with

    alcohol briefly

    about 20 sec

    Counterstain with

    safranin for 12 min

    G+

    G-

    Step 2

    Step 3

    Step 4

  • 2.2 Improving Contrast in Light Microscopy 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

  • 2.2 Improving Contrast in Light Microscopy 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 samples

    bright-field fluorescence DAPI-stained

  • 2.3 Imaging Cells in Three Dimensions

    Differential Interference Contrast (DIC) Microscopy Uses a polarizer to create two distinct beams

    of polarized light

    Gives structures such as endospores, vacuoles, and granules a three-dimensional appearance

    Structures not visible using bright-field microscopy are sometimes visible using DIC

  • 2.3 Imaging Cells in Three Dimensions Confocal Scanning Laser Microscopy (CSLM)

    Uses a computerized microscope coupled with a laser source to generate a three-dimensional image

    Computer can focus the laser on single layers of the specimen

    Different layers can then be compiled for a three-dimensional image

    Resolution is 0.1 m for CSLM

  • 2.4 Electron Microscopy Electron microscopes use electrons instead

    of photons to image cells and structures

    Two types of electron microscopes:

    Transmission electron microscopes (TEM)

    Scanning electron microscopes (SEM)

    Electronsource

    Evacuatedchamber

    Sampleport

    Viewingscreen

  • Cytoplasmic membrane

    DNA(nucleoid)

    Septum Cell wall

    2.4 Electron Microscopy

    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 (2060 nm) and

    stained

    Animation: Electron Microscopy

  • 2.4 Electron Microscopy

    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 15100,000

  • II. Cells of Bacteria and Archaea

    2.5 Cell Morphology

    2.6 Cell Size and the Significance of Being Small

  • 2.5 Cell Morphology

    Morphology = cell shape

    Major cell morphologies

    Coccus (pl. cocci): spherical or ovoid

    Rod: cylindrical shape

    Spirillum: spiral shape

    Cells with unusual shapes

    Spirochetes, appendaged bacteria, and filamentous bacteria

    Many variations on basic morphological types

  • Coccus

    Rod

    Spirillum

    Spirochete

    Stalk Hypha

    Budding and

    appendaged bacteria

    Filamentous bacteria

  • 2.5 Cell Morphology

    Morphology typically does not predict physiology, ecology, phylogeny, etc. of a prokaryotic cell

    Selective forces may be involved in setting the morphology

    Optimization for nutrient uptake (small cells and those with high surface-to-volume ratio)

    Swimming motility in viscous environments or near surfaces (helical or spiral-shaped cells)

    Gliding motility (filamentous bacteria)

  • 2.6 Cell Size and the Significance of Being Small

    Size range for prokaryotes: 0.2 m to >700 m in diameter Most cultured rod-shaped

    bacteria are between 0.5 and 4.0 m wide and 200 m in diameter

  • r = 1 m

    r = 2 m

    r = 1 m

    r = 2 m

    Surface area (4r2) = 12.6 m2

    Volume ( r3) = 4.2 m3

    Surface area = 50.3 m2

    Volume = 33.5 m3

    34

    SurfaceVolume

    = 3

    SurfaceVolume

    = 1.5

    2.6 Cell Size and the Significance of Being Small

    Surface-to-Volume Ratios, Growth Rates, and Evolution Advantages to being

    small Small cells have more

    surface area relative to cell volume than large cells (i.e., higher S/V)

    Support greater nutrient exchange per unit cell volume

    Tend to grow faster than larger cells

  • 2.6 Cell Size and the Significance of Being Small

    Lower Limits of Cell Size Cellular organisms < 0.15 m in diameter are

    unlikely Open oceans tend to contain small cells (0.20.4

    m in diameter)

  • II. The Cytoplasmic Membrane and Transport

    2.7 Membrane Structure

    2.8 Membrane Functions

    2.9 Nutrient Transport

  • 2.7 Membrane Structure

    Cytoplasmic membrane:

    Thin structure that surrounds the cell

    68 nm thick

    Vital barrier that separates cytoplasm from environment

    Highly selective permeable barrier

    Allows concentration of specific metaboli