Chapter 3 Microscopy and Cell structure

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					         Chapter 3
Microscopy and Cell structure
          Biology 261
          Prof. Santos
           Fall 2011
• Microscope- tool used to see objects too small
  to be seen by the naked eye. Types of
  microscopes used in the study of
  microorganisms include the light microscope,
  dark field microscope, phase contrast
  microscope, confocal microscope, interference
  microscope, fluorescence microscope, electron
  microscope, and atomic force microscope.
             Terms to know
• Bright field microscopy- lights rays are used to
  evenly illuminate the field of view.
• Dark field microscopy- light is directed
  towards the specimen at an angle. This makes
  it possible for the unstained specimen to
  appear more visible against a dark background.
• Contrast- basically it is the number of visible
  shades in a specimen.
• Microscopes that increase contrast include,
  phase contrast microscope, interference
  microscope, dark field microscope,
  fluorescence, and confocal microscope.
           Light Microscope
1-Light microscope- uses a beam of light to
  create an enlarged image of the specimen. The
  light microscope can either use a mirror or a
  light bulb to pass light through the specimen.
  You can magnify a specimen up to 1000x with
  a good light microscope
      Parts of the microscope
A- body tube- connects the eyepiece with the
 rotating nosepiece.

B-eye piece- the lens the observer looks through.
C-rotating nosepiece- holds the objectives and
  allows you to switch from one to the other.
D-objective lens- the lens found on the nosepiece
  that magnifies the image. Most microscopes
  contain three; the low power, high power, and
  the oil immersion lens.
• *To determine the total magnification power,
  multiply the power of the eyepiece with the
  power of the objective lens. For example; if
  the eyepiece is 10x and you are using the high
  power, which is 40x, then the total magnifying
  power is 400x. So, it means you are looking at
  the object 400x closer.
E-coarse adjustment knob-allows you to move the stage
  closer to the objective lens and focus the specimen.
  When you first place a slide in the scope, you should
  always start with low power and focus with the
  coarse adjustment.
F-fine adjustment knob-allows you to move the stage
  very slowly and finely focus the specimen under high
  power. Keep in mind that when you move to high
  power; the amount of light passing through will
  diminish. You might need to adjust the diaphragm.
G-stage- broad flat platform where you place the
 slide.
H-stage clips- hold the slide in place.
I-diaphragm- a circular flat wheel underneath the
  stage that allows you to control the amount of
  light passing through.
J-arm- used to carry the microscope and for
  support.
K-light source- provides the light needed to
 create an enlarged image of specimen. The
 light source is either a mirror or a light bulb.
Resolution- the ability of a microscope to
 separate and show two points that are very
 close together.
Resolution depends on the type of lens used,
 wavelength of the light, magnification, and
 specimen preparation.
Resolution can be expressed as
d= .5 (wavelength)/ NA

d= distance between the two points
NA = numerical aperture, a mathematical
  expression that describes how well the
  condenser directs light rays through the
  specimen
     To increase resolution
1-

2-

3-
• The maximum resolving power of the best
  light microscope is .2 um due to the
  wavelength of light.
• When using the 100x objective lens, one must
  use immersion oil to maximize resolution. This
  is because of the difference in the refractive
  index of air and glass. As light rays pass from
  one medium to another, light rays will bend if
  there is a difference in the refractive index.
• Refractive index is the velocity of light as it
  travels through a medium.
        Electron Microscope
Electron microscope- uses a beam of electrons
to create an enlarged image of the specimen.
An electron microscope can magnify a
specimen up to 250,000x closer. The
wavelength used is less than the wavelength of
light, thus the resolution is greater.
                     2 types
• TEM- transmission electron microscope allows
  one to see fine detail structures in side the cell.

• SEM- scanning electron microscope allows
  one to see the surface details of cells like the
  membrane proteins.
3-Phase contrast microscope- allows you
to see unstained cells by altering the
background of the cell. This microscope
has a device that allows it to amplifies
differences in refractive index to create a
contrast.
4-fluorescence microscope
    Projects ultra violet light causing fluorescent
  molecules in the specimen to emit a longer
  wavelength.

5- Confocal microscope
   Mirrors are used to scan a laser beam across
 successive regions and planes of a specimen. A
 computer program constructs a 3D image.
6- Interference microscope causes the
  specimen to appear as a 3D image. The
  most common one is the Nomaski
  differential interference microscope. This
  microscope has a special device that
  separates light going through a specimen
  into 2 beams and then recombines them.
  The light rays are out of phase when they
  recombine, yielding a 3D image.
7- Dark field microscope
   this type of light microscope has a special
  device that directs light at an angle so that only
  light scattered by the specimen enters the
  objective so one sees the specimen against a
  dark background.
8- Atomic force microscope
   This very powerful microscope produces a
  very detailed image of the surface of an
  specimen by using a very sharp probe (stylus)
  to move across the surface and “feel” the
  bumps and valleys of the atoms of the surface.
                   Staining
• Use of either a basic or acidic dye to color
  certain cell parts and make it more visible to
  the naked eye. Three types of staining
  techniques; simple, differential and special
  stains for the flagella, spores, and capsule.
                      Dyes
• Basic dyes carry a positive charge. They stain
  the negative parts of a cell like proteins and
  nucleic acids. Examples include methylene
  blue, crystal violet, safranin, malachite green.
• Acidic dyes carry a negative charge. They are
  usually used to stain the background to
  observe colorless cells.
• Neutral no charge
• Staining allows you to see parts of the cell you
  would not ordinarily see. Cells are clear! By
  adding a stain you are basically coloring
  certain parts of the cell that take in the dye.
              Prokaryotic cell
 Morphology
Three basic shapes; spherical called coccus,
     cylindrical called bacillus and spiral. There
     are variations!
coccobacillus- short rod
vibrio- a short curve rod
spirochete- a long helical cell with a flexible cell
     wall and unique mode of utility
Pleomorphic- bacteria that vary their shape
Grouping
       Multicellular association
1- fruiting body

2- biofilm
     Structure of prokaryote cell
1-Flagella provides motility. The flagella is
  made up of three basic parts, filament, hook
  and basal body.
2-Pili proteins that enable the bacteruim to
  adhere to surfaces. Fimbriae allow bacteria to
  adhere to surfaces and sex pili allow DNA
  transfer between bacteria.
3-The capsule a viscous and gelatinous layer that
  surrounds bacteria. It enables bacteria to
  adhere to certain surfaces and allows
  organisms to avoid innate defense systems and
  cause diseases. Ex, Streptococcus pneumoniae.
4- slime layer- gel like layer that is diffuse and
  irregular. This layer is composed of
  polysaccharides and enables the bacteria to
  adhere to surfaces and grow as biofilm.
Ex; Streptococcus mutans grows as biofilm on
  your teeth to form dental plaque.
5- The cell wall a rigid covering consisting of
  peptidoglycan that gives the bacterium its
  shape and protection.
• The type of cell wall distinguishes between 2
  types of bacteria; gram negative and gram
  positive.
• Peptidoglycan is a macromolecule found only
  in bacteria.
• The basic structure of peptidoglycan is an
  alternating series of 2 major subunits, N-
  acetylmuramic acid (NAM) and N-
  acetylglucosamine (NAG). These subunits are
  covalently bonded to each other to form a
  glycan chain.
• Attached to each NAM molecule is a string
  of 4 amino acids, a tetrapeptide chain.
  Cross linkages can form between adjacent
  chains thus joining adjacent glycan chains.
• In gram negative bacteria these tetrapeptides
  are joined directly.
• In gram positive bacteria they are usually
  joined indirectly by a peptide interbridge.
• In gram positive bacteria the peptidoglycan
  layer is thick.
• In gram negative bacteria the peptidoglycan
  layer is thinner.
               Teichoic acid
• In gram positive bacteria there are polymers of
  teichoic acid present. These teichoic acid
  polymers are covalently linked to the NAM
  molecules of the glycan chain.
• These polymers consists of chemically
  modified ribose or glycerol sugars connected
  by phosphates. Sugars and D- alanine may be
  attached to these polymers providing antigenic
  determination.
• Teichoic acid provides rigidity to the cell wall.
            Outer membrane
• In gram negative bacteria there is an outer
  membrane outside the peptidoglycan. It is a
  unique lipid bilayer.
• The outer membrane is unlike any other
  membrane. The outside leaflet consists of
  Lipopolysaccharides instead of phospholipids.
• The outer membrane is sometimes called the LPS or
   lipopolysaccharide layer.
• The outer membrane is joined to peptidogylcan by
   means of lipoproteins
• Two parts are importance for medical reasons;
a- Lipid A is the portion that anchors the LPS in the
   lipid bilayer. It plays a role in the immune system.
b- O specific polysaccharide is a chain of sugar
   molecules opposite the Lipid A. Allows for
   identification.
Gram positive
                  periplasm
• In gram negative bacteria, it is the space
  between the inner and outer membrane.
            Intracellular parts
6- Bacterial chromosome is usually circular
  double stranded molecule located in a region
  called the nucleoid
7- Plasmids are small, supercoiled, circular
  double stranded pieces of DNA that contain
  few genes.
8- Endospore is a type of dormant cell that
  resists harsh conditions.
9- Cytoskeleton proteins that provide support.
10- Gas vesicles provide buoyancy
11- granules are accumulations of substances produced
  in excess.
• Examples are glycogen, poly beta hydroxybutyrate,
  and volutin.
• Volutin- a storage form of phosphate. They stain red
  with methylene blue, sometimes called
  metachromatic granules. Role is unclear. Thought to
  be involved in energy storage and pH balance inside
  the cell.

12- ribosomes involved in protein production
             Eukaryotic cell
1- Plasma membrane consisting of asymmetrical
  lipid bilayer.
The cell membrane consists of proteins and
  lipids.
          Internal protein parts
2- Cilia/flagella are protein structures that
  consists of microtubules in a 9 +2
  arrangement. Cilia and flagella function in
  motility.
3- cytoskeleton consists of proteins such as
  microtubules, actin filaments and intermediate
  filaments that function in cell structure/support
  and act as a molecular monorail.
4- ribosomes are involved in protein production.
5- Chloroplast- double membrane bound
  organelle involved in photosynthesis
6- Endoplasmic reticulum is a system of canals
  involved in the production of macromolecules
  destined to be secreted to other organelles or
  outside. Two types, smooth and rough.
7- Golgi Apparatus is a system of flat
  membranes involved in the modification of
  material made in the ER. The vesicles are
  coated with special carbohydrates and
  phosphate groups that signal the vesicles to
  their specific location.
9- Nucleus is the control center of the cell.
  Contains the genetic material and is
  surrounded by a nuclear envelope.
10- Mitochondrium is the site of cellular
  respiration
11- Lysosomes
12- peroxisomes are organelles us to oxidize
  substances, breaking down lipids, and
  detoxifying certain chemicals.

				
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