Introduction to Microbiology

Clinical Focus: Use of microscopy and staining to diagnose infections

1. Case study "Cindy"

2. Case studies: Meningococcal meningitis

A. Properties of light and lenses

• Wavelength and energy
• Reflection, absorbance, and transmittance
• Color: absorption or reflection of different wavelengths
• Fluorescence and phosphorescence
• Lenses: refraction (bending) of light   
  • Magnification: eyepiece 10X, objective lens ranges from 4X to 100X
  • Resolution: function of refractive index (typically 0.4 um at best)

B. Types of microscopes

1. Light microscopes
   • Bright field
   • dark field
   • phase contrast
   • fluorescence
   • confocal
   • Differential interference contrast
   • two-photon
2. Electron microscopes
   • scanning EM
   • transmission EM
3. Scanning probe microscopes
   • Atomic Force
   • Scanning Tunneling

C. Staining and contrast

1. Some microbes are big enough to look darker than the background without staining
   • algae, fungi, protozoa, large bacteria
   • Clinical samples (human cells, eukaryotes, parasites)
2. Most bacteria are too thin to absorb light; must be stained to be seen with a bright field microscope
3. Other techniques are available to view unstained bacteria & microbes
   • Dark field
   • Phase contrast
4. Viruses cannot be seen without EM and staining + extensive preparation
   • Not practical in a hospital setting
   • molecular/genetic testing usually required to ID viruses

D. Types of stains

1. Direct stains (basic stains)
   • Crystal violet
   • Safranin
   • Methylene blue
2. Indirect stains (negative stains)
3. Differential stains
   • Gram stain
   • Acid-fast stain
4. Structure-specific stains
   • capsule stain
   • endospore stain
   • flagella stain
5. Stains for protozoa
   • Giemsa: Trichomonas
   • Trichrome: Giardia
   • Ziehl-Neelson: Cryptosporidia (cysts)

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