The Study of Microbial Structure:

1

• The Study of Microbial Structure
• Microscopy and Specimen Preparation

2
(No Transcript)
3
Lenses and the Bending of Light

• light is refracted (bent) when passing from one
  medium to another
• refractive index
• a measure of how greatly a substance slows the
  velocity of light
• direction and magnitude of bending is determined
  by the refractive indexes of the two media
  forming the interface

4
Lenses

• focus light rays at a specific place called the
  focal point
• distance between center of lens and focal point
  is the focal length
• strength of lens related to focal length
• short focal length ?more magnification

5
(No Transcript)
6
Types of light microscopes

• Bright-field most widely used, specimen is
  darker than surrounding field
• Dark-field brightly illuminated specimens
  surrounded by dark field
• Phase-contrast transforms subtle changes in
  light waves passing through the specimen into
  differences in light intensity, best for
  observing intracellular structures

7
3 views of a cell
8
Fluorescence Microscope

• Modified compound microscope with an ultraviolet
  radiation source and a filter that protects the
  viewers eye
• Uses dyes that emit visible light when bombarded
  with shorter uv rays.
• Useful in diagnosing infections

9
(No Transcript)
10
Electron microscopy

• Forms an image with a beam of electrons that can
  be made to travel in wavelike patterns when
  accelerated to high speeds.
• Electron waves are 100,000X shorter than the
  waves of visible light.
• Electrons have tremendous power to resolve minute
  structures because resolving power is a function
  of wavelength.
• Magnification between 5,000X and 1,000,000X

11
2 types of electron microscopes

• Transmission electron microscopes (TEM)
  transmits electrons through the specimen darker
  areas represent thicker, denser parts and lighter
  areas indicate more transparent, less dense parts
• Scanning electron microscopes (SEM) provides
  detailed three-dimensional view. SEM bombards
  surface of a whole, metal-coated specimen with
  electrons while scanning back and forth over it.

12
Transmission Electron Micrograph
13
Scanning Electron Micrograph
14
Newer Techniques in Microscopy

• confocal microscopy and scanning probe microscopy
• have extremely high resolution
• can be used to observe individual atoms

15
Preparation and Staining of Specimens

• increases visibility of specimen
• accentuates specific morphological features
• preserves specimens

16
Fixation

• process by which internal and external structures
  are preserved and fixed in position.
• process by which organism is killed and firmly
  attached to microscope slide
• heat fixing
• preserves overall morphology but not internal
  structures
• chemical fixing
• protects fine cellular substructure and
  morphology of larger, more delicate organisms

17
Dyes and Staining

• dyes
• make internal and external structures of cell
  more visible by increasing contrast with
  background
• have two common features
• chromophore groups
• chemical groups with conjugated double bonds
• give dye its color
• ability to bind cells

18

• simple staining
• a single staining agent is used
• basic dyes are frequently used
• dyes with positive charges
• e.g., crystal violet

19
Differential Staining

• divides microorganisms into groups based on their
  staining properties
• e.g., Gram stain
• e.g., acid-fast stain

20
Gram staining

• most widely used differential staining procedure
• divides Bacteria into two groups based on
  differences in cell wall structure

21
primary stain
mordant
decolorization
counterstain
positive negative
22
Escherichia coli a gram-negative rod
23
Acid-fast staining

• particularly useful for staining members of the
  genus Mycobacterium
• e.g., Mycobacterium tuberculosis causes
  tuberculosis
• e.g., Mycobacterium leprae causes leprosy
• high lipid content in cell walls is responsible
  for their staining characteristics

24
Staining Specific Structures

• Negative staining
• often used to visualize capsules surrounding
  bacteria
• capsules are colorless against a stained
  background

25

• Spore staining
• double staining technique
• bacterial endospore is one color and vegetative
  cell is a different color
• Flagella staining
• mordant applied to increase thickness of flagella