Lecture Notes on Microscopy in Microbiology

Introduction to Microscopy

• Essential in microbiology for visualizing very small microbes.
• Practice with microscopes, especially under oil immersion, is key.
• Microscopy: Making small things visible to the human eye.

Importance of Oil Immersion

• Necessary to see individual cells.
• Measurements are often in the micrometer range (1mm = 1000 micrometers).

Key Concepts in Microscopy

• Magnification vs. Resolution
  • Magnification: Enlarges objects.
  • Resolution: Ability to distinguish two separate objects.
• High-resolution microscopes provide clarity and are typically expensive.
• Importance of light passing through samples for good resolution.

Types of Microscopes

• Compound Light Microscope: Used to see eukaryotic cells, bacteria at high power with oil immersion.
• Electron Microscopes: Used for viruses; two types:
  • Transmission Electron Microscope (TEM)
  • Scanning Electron Microscope (SEM)
• Fluorescence Microscopy: Uses UV light for diagnosis.
• Other advanced microscopy techniques include Scanning Tunneling Microscopy and Atomic Force Microscopy.

Preparing Samples for Microscopy

• Making Smears: Essential to practice making consistent smears for light transmission.
• Heat Fixing: Kills organisms, adheres them to slides, and opens cell walls.
• Stains:
  • Cationic Stains: Positively charged and adhere well to bacterial cell walls.
  • Anionic Stains: Not commonly used as they do not adhere well.

Types of Stains

• Simple Stain: Uses one dye to show shape and arrangement.
• Differential Stain: Provides more information by showing differences (e.g., Gram stain, Acid-fast stain, Spore stain).
  • Gram-positive vs. Gram-negative differences.
• Negative Stain: For visualizing capsules.
• Flagella Stain: Visualizes bacterial tails.
• Spore Stain: Identifies endospores vs. vegetative cells.

Practical Application

• We will be using compound light microscopes under bright field conditions.
• Practice making slides and using stains to see bacteria.
• Recognize the difference between magnification and resolution during lab practice.