microscope

Microscopy & the Cell

Course Information

• Instructor: Dr. Kasumu

• Course Title: MICROBIOLOGY (MBE 250/MBP 250)

Learning Objectives

• Differentiate between prokaryotic and eukaryotic cells

• Understand the functions of various cell components

• Comprehend the function of different microscopes

• List the various types of microscopes

What is a Cell?

• Definition:

  • The basic structural, functional, and biological unit of all living organisms.

  • Metabolic Processes: Associated with life occur within cells.

  • Cell Origin: Cells arise from pre-existing cells through cell division.

  • Genetic Material: Cells contain hereditary material that is passed on to daughter cells during division.

Types of Cells

Prokaryotic Cell

• Characteristics:

  • Lack membrane-bound organelles (e.g., mitochondria, Golgi bodies).

  • No true nucleus.

  • Mostly unicellular organisms (e.g., bacterial species).

Generalized Structure of a Prokaryote

• Components:

  • DNA strand

  • Plasmid

  • Nucleoid region

  • Ribosome

  • Cytosol

  • Capsule

  • Cell wall

  • Plasma membrane

  • Fimbriae

  • Flagellum

Eukaryotic Cell

• Characteristics:

  • Contains several internal structures (organelles).

  • Enveloped nucleus.

  • Can be unicellular or multicellular.

  • Unicellular Example: Yeast

  • Multicellular Examples: Plants and animals

Generalized Structure of a Eukaryote

• Components:

  • Endoplasmic reticulum

  • Cytoskeleton

  • Cytoplasm

  • Ribosomes

  • Nucleus

  • Mitochondrion

  • Lysosome

  • Golgi body

Principal Differences Between Prokaryotic and Eukaryotic Cells

Prokaryotic Cell Division: Binary Fission

1. Cell elongation and DNA replication occurs.

2. Cell wall and plasma membrane begin to divide.

3. Forms a cross-wall that completely surrounds divided DNA.

4. Cells separate.

Microscopy Overview

• Use of Microscopy:

  • Initial detection and definitive identification of microbes.

  • Helps in identifying the morphological properties of an organism.

Microscopic Methods

• Five General Methods:

  1. Brightfield (light) microscopy

  2. Darkfield microscopy

  3. Phase contrast microscopy

  4. Fluorescence microscopy

  5. Electron microscopy

Brightfield (Light) Microscopy

• Components:

  • Light source, specimen stage, condenser, two lens system.

• Visualization: Specimen is visualized by transillumination. Requires staining with dye for improved resolution.

Components of a Brightfield Microscope

• Light Source: Illuminates the specimen.

• Condenser: Focuses light on the specimen.

• Objective and Ocular Lens: Magnify the image of the specimen; oil immersion may be used to reduce light dispersion.

Advantages and Limitations of Brightfield Microscopy

• Advantages:

  • Cost-effective and suitable for resource-limited settings.

• Limitations:

  • Low resolving power and requires stains for improved resolution.

Darkfield Microscopy

• Operation:

  • Similar objective and ocular lenses to brightfield; uses special condenser to prevent direct illumination of the specimen, allowing only oblique light to reach it.

  • Specimens appear brightly illuminated against a black background, improving resolving power.

Advantages and Limitations of Darkfield Microscopy

• Advantages:

  • Simple and effective for unstained biological samples.

• Limitations:

  • Requires strong illumination, which may damage samples.

Phase Contrast Microscopy

• Functionality:

  • Examines internal details of microbes by affecting parallel light beams based on specimen density, creating a 3D image.

Fluorescence Microscopy

• Process:

  • Involves staining microbes with fluorescent dyes and examining them under a fluorescence microscope using shorter wavelength light.

• Drawbacks:

  • Higher setup and operational costs.

Electron Microscopy (EM)

• Capabilities:

  • High resolving power, approximately 1000 times that of light microscopes.

  • Utilizes a beam of electrons focused with electromagnetic lenses.

• Staining:

  • Heavy metals (e.g., gold and osmium tetroxide) are commonly used.

• Limitations:

  • Cannot view living specimens.

Comparing Microscopic Resolution

• Human Eye: Distinguishes objects down to a fraction of a millimeter.

• Light and Electron Microscopes: Can visualize down to an angstrom, including cells, bacteria, single molecules, or atoms.

Staining Methods

• Purpose: Enhances visibility of cellular structures under a microscope.

• Application: Different stains preferentially target various cell components (e.g., nucleus, cell wall).

Preparation of Slides

1. Permeabilization:

   • Treatment with mild surfactant to dissolve cell membranes, allowing larger dye molecules to enter.

2. Fixation:

   • Aims to preserve cell or tissue morphology through chemicals or heat.

3. Mounting:

   • Attaching samples to microscope slides for observation; cells can be grown directly or applied.

4. Staining:

   • Immersing samples in dye solutions after fixation or mounting, followed by rinsing and observation.

Commonly Used Stains

1. Differential Stains:

   • Gram Stain:

     • Most commonly used in microbiology to separate Gram-positive and Gram-negative bacteria based on cell wall characteristics.

     • Visual Examples: Gram-positive cocci (e.g., S. aureus) and Gram-negative bacilli (e.g., E. coli).

2. Acid-Fast Stains:

   • Ziel-Neelsen Stain: Used for Mycobacteria and other acid-fast organisms.

   • Auramine-Rhodamine: Fluorescent dyes for acid-fast organisms.

   • Modified Acid-Fast Stain: Used for weak acid-fast organisms (e.g., Nocardia spp.).

Further Reading

• Recommended Texts:

  • Murray: Medical Microbiology (6th edition, ch. 14 on microscopic principles and applications).

  • Mims: Medical Microbiology (4th ed., ch. 32 on the diagnosis of infection focusing on non-cultural techniques for laboratory diagnosis).