Lecture 3: Microscopy & Microbial Diversity

Lecture 3: Microscopy & Microbial Diversity

• Lecture covers microscopy techniques and introduces microbial diversity.
• Previous lecture (Lecture 2) discussed culturing microbes, difficulties in working with them, sterile growth media, handling microorganisms (inoculation, incubation, isolation, inspection, identification), disposal of cultures, disinfectants, and antiseptics.

Learning Outcomes

• Light Microscopy:
  • Preparing bacterial cells for microscopy
  • Light microscope resolution
• Electron Microscopy:
  • Scanning Electron Microscope (SEM) used for observing external cell features
  • Transmission Electron Microscope (TEM) used for observing internal cell structures.
• Domains of Life:
  • Types of microorganisms (prokaryotic, eukaryotic, non-cellular)
• Cell Structure:
  • Eukaryotic cell structure
  • Prokaryotic cell structure
  • Comparison of eukaryotic and prokaryotic cells

Light Microscopy Details

• Magnification: How much an image is enlarged under a microscope. It can be increased without limit.
• Resolution: The amount of detail you can see in an image under a microscope. Increasing magnification does not improve resolution. Light microscope resolution is limited to approximately 0.2 µm.
• Resolution Limit: Limited by the wavelength of light. Any two lines closer together than 0.275 micrometers will appear as a single line.

Light Microscope Parts

• Ocular: Eyepiece
• Objective: Lenses
• Stage: Holds the specimen
• Condenser: Focuses light on the specimen
• Focusing Knobs: Used to adjust focus
• Light Source: Illuminates the specimen

Preparing Bacterial Cells for Microscopy

• Spread culture: Spread a thin film of the culture on a slide.
• Dry in air: Allow the culture to dry in air
• Pass slide through flame: To fix the sample
• Flood with stain: Stain the specimen
• Rinse and dry: Rinse the slide and allow it to dry

Resolution (Light Microscopy)

• Light microscopes cannot see details smaller than half the wavelength of light (about 0.275 µm).

Electron Microscopy

• Electron Microscopy I: Uses electrons instead of light, providing much higher resolution (>100000 x greater than a light microscope). Electrons have a much smaller wavelength. The system needs to be kept a vacuum.
• Electron Microscopy II: Electron beams are focused on the sample and absorbed or scattered by its components.
• Electron Microscopy III:
  • SEM: Scans the surface of the sample looking at external features, coating the sample in a metal such as gold. The sample interacts with electrons and scattered electrons create the image.
  • TEM: Examines internal structures of very thin sections of the sample because electrons do not penetrate a thick sample.

Domains of Life

• Prokaryotes: Bacteria and Archaea
• Eukaryotes: Fungi, protists, plants, and animals
• Non-cellular: Viruses and Prions

Eukaryotic Versus Prokaryotic Cells

• Comparison chart: covered in the presentation detailing differences in structure, functions, and other aspects such as cell wall composition.

  • Prokaryotes do not have a nucleus or internal organelles (like mitochondria, chloroplasts).
  • Eukaryotes have a nucleus and several internal organelles to carry out different functions.
  • Organelles like mitochondria are described in eukaryotes, but are missing in prokaryotes.

• Respiratory enzymes and electron transport chains: Located in mitochondria (eukaryotes) or the cytoplasmic membrane (prokaryotes).

• Cell wall: Eukaryotic cell walls can be various materials without peptidoglycan, prokaryotic cell walls have peptidoglycans.

• Locomotion organelles: Flagella in both prokaryotes and eukaryotes (but differ in structure). Cilia present in eukaryotes.

This lecture delves into microscopy techniques and the rich diversity of microorganisms. Learn about light and electron microscopy methods, cell structures of different life forms, and the classification of microbes. Understand the significant differences between prokaryotic and eukaryotic cells as well as non-cellular entities.