CHAP 2: Methods in Microbiology

• Electron Microscope was discovered

  1940
• Units of measurement:

  1 micrometer (μm) = 10^-6 m

  1 nanometer (nm) = 10^-9 m
• characteristics of a good microscope

  adequate magnifying power

  high resolving power

  provide good contrast

  serves your purpose
• TYPES OF MICROSCOPES

  light microscopes

  electron microscopes
• uses light waves and mirrors

  LIGHT MICROSCOPES
• short focal length

  only 1 lens

  magnification: 300x

  Simple Microscope
• 2 sets of lenses, magnification: 1000x


  
  

  Compound or Complex
• use electron beams as energy source

  has higher magnification and resolving power

  for objects smaller than 0.2 mm in diameter

  in vacuum


  ELECTRON MICROSCOPES
• TYPES OF LIGHT MICROSCOPES (7)

  BRIGHT FIELD

  DARK FIELD

  PHASE CONTRAST

  DIFFERENTIAL INTERFERENCE CONTRAST

  FLUORESCENCE

  CONFOCAL

  TWO-PHOTON
• • objects under study are darker

  • microscopic field is brightly lit

  • gross morphology


  BRIGHT FIELD
• • microscopic field is dark

  • objects under study are luminous

  • invisible specimen in the ordinary light microscope

  • specimen cannot be stained by standard methods

  • specimen distorted by staining


  DARK FIELD
• • principle is based on variations in the refractive indices (measure of bending or refracting of a beam of light on entering a denser medium)

  • not necessary to fix or stain cells

  • detailed examination of internal structure

  PHASE CONTRAST
• • principle is based on variations in the refractive indices

  • advantage: no diffraction halo associated with phase contrast

  • disadvantage: the three-dimensional appearance may not represent reality


  DIFFERENTIAL INTERFERENCE CONTRAST
• • makes use of fluorochromes

  • visualizes specimens that fluoresce

  • detection of immunological reactions


  FLUORESCENCE
• INDIRECT IMMUNOFLUORESCENCE 


  fluorochrome-secondary antibody-primary antibody-antigen
• DIRECT IMMUNOFLUORESCENCE

  fluorochrome-primary antibody-antigen
• • useful for thick specimens like biofilms

  • used to visualize structures


  CONFOCAL
• • useful for examining living cells within intact tissues

  • currently limited to advanced clinical and research laboratories

  TWO-PHOTON
• Types of Electron Microscopes (2)

  TRANSMISSION ELECTRON MICROSCOPE (TEM)

  SCANNING ELECTRON MICROSCOPE (SEM)
• • ultrastructure in thin section of the cells

  • can project images in a much higher resolution—up to the atomic level of thinner objects

  • examine viruses


  TRANSMISSION ELECTRON MICROSCOPE (TEM)
• • surface features of viruses and cells

  • reveals a 3-D image


  SCANNING ELECTRON MICROSCOPE (SEM)
• Scanning Probe Microscope (2)


  SCANNING TUNNELING MICROSCOPE (STM) eg: pure gold surface

  ATOMIC FORCE MICROSCOPE (AFM) eg: nanocellulose
• EXAMINATION OF MICROORGANISMS (2)

  Living or Natural State

  Stained Preparations
• • observation of unaltered/undistorted characteristics

  • cellular processes can be studied

  • motility can be observed

  • simple to prepare


  Advantages of Living or Natural State Examination
• • refractive index of cells almost similar to that of water


  Disadvantage of Living or Natural State Examination
• • provides contrast

  • slides can be preserved

  • specimens are killed


  Advantages of Stained Preparations
• • more complicated and tedious to prepare

  • expensive


  Disadvantages of Stained Preparations
• Three basic steps in staining microorganisms:

  Smear Preparation

  Fixation

  Staining
• a thin, dry film of microorganisms

  Smear
• • kills the cells

  • makes the cells sticky

  • increases apparent diameter of cells


  Fixation
• Types of Fixation (2)

  Heat Fixation

  Chemical Fixation
• through direct flame or steam

  Heat Fixation
• fixation using alcohols

  Chemical Fixation
• application of biological dyes

  Staining
• • organic compounds carrying chromophoric ions

  • make cell’s internal and external structures more visible with the increased contrast with the background

  Dyes (Stains)
• Types of Stains (3)

  Basic or Positively-Charged Dye

  Acidic or Negatively-Charged Dye

  Neutral
• MAJOR STAINING PROCEDURES (3)

  SIMPLE STAINING

  DIFFERENTIAL STAINING

  STRUCTURAL STAINING
• only one dye stain

  SIMPLE STAINING
• TYPES OF SIMPLE STAINING

  (1) Positive / Direct

  • cells are the same color as the dye

  • crystal violet, malachite green, methylene blue, safranin

  (2) Negative / Indirect

  • cells are colorless or luminous

  • acid fuchsin, eosin, rose bengal, india ink, nigrosin
• two or more dyes and/or reagents staining

  DIFFERENTIAL STAINING