Chapter 2

• Microbe sizes range from μm to nm
• Resolving Power (Resolution) is the distance between 2 points that can be distinguished
• Light Properties:

  • Wavelength is part of the electromagnetic spectrum
  • Shorter wavelength means higher energy and more resolution
• Light Travel:

  • Reflection: waves are bounced off some materials
  • Transmission: waves pass through
  • Absorption: energy modification
  • Refraction: altered angle of movement
• Compound Light Microscopy components:

  • Condenser
  • Iris diaphragm
  • Objective lenses
  • Ocular lens(es)
  • Stage
  • Focusing knobs
  • Total Magnification
• Light Microscopy Types:

  • Bright Field
  • Dark Field, used for Spirochaetes like T. pallidum and B. burgdorferi
• Dual Beam Microscopy:

  • Uses 2 light beams
  • Phase Contrast creates areas of contrast through interference for internal cellular structures
  • Nomarski provides higher resolution and shallow depth of field
• Fluorescence Microscopy:

  • Uses ultraviolet light to excite electrons that emit light after UV absorption
  • Contrasts natural fluorescence versus fluorochromes
  • FAb staining involves antibodies tagged with fluorescent dyes specific for molecules
• Imaging Techniques:

  • Confocal Microscopy (UV)
  • Digital Microscopy
• Electron Microscopy (EM):

  • Uses X-rays focused by electromagnets
  • Transmission Electron Microscopy (TEM) uses thin tissue slices and heavy metal staining
  • Scanning Electron Microscopy (SEM) involves gold/carbon coating for profiles or fractures
• Light Microscope Specimen Preparation:

  • Wet Mounts: thick liquid preparation
  • Smears: placement of cells, air drying, heat fixation
• Staining Principles:

  • Acidic (-ve) / Basic (+ve)
  • Simple Stains
  • Differential Stains
  • Special Stains
• Gram Stain:

  • Developed by Hans Gram
  • Distinguishes microbes based on peptidoglycan content in cell wall
  • CV-I complex involves crystal violet + iodine forming a large molecular complex
  • Alcohol wash differentiates Gram +ve and Gram -ve cells
  • Saffranin counterstain distinguishes cell types
  • Diagnostic importance in targeting peptidoglycan with antibiotics like Penicillins, Monobactams, and Carbapenems
• Ziehl-Neelsen Acid Fast Stain:

  • Used for Acid Fast Bacteria like Mycobacterium genus
  • Identifies tuberculosis and leprosy through waxy lipid mycolic acid repelling most dyes
• Negative (Capsule) Stain:

  • Visualizes the Capsule with thick glycocalyx around the cell
  • Halo effect seen due to negatively charged dyes repelled by the capsule
  • Virulence factor that repels phagocytic WBC and may prevent antibiotic entry into cells
• Endospore Stain (Schaefer-Fulton):

  • Endospores are visualized for pathogens like Clostridium tetani, C. botulinum, C. perfringens, and Bacillus anthracis
  • Endospores are resistant to heat and dessication
• What is SEM?

  Scanning electron microscope
• What is TEM?

  Transmission electron microscope