Microscopy, Staining, and Classification in Microbiology

Microscopy, Staining, and Classification

Overview

This study guide covers fundamental concepts in microbiology related to microscopy, staining techniques, and the classification of microorganisms. Understanding these topics is essential for identifying and studying microbes in laboratory and clinical settings.

Microscopy

Units of Measurement in Microbiology

• Nanometer (nm): $10^{-9}$ meters. Used to measure viruses and subcellular structures.

• Micrometer (μm): $10^{-6}$ meters. Used for bacteria and most cells.

• Millimeter (mm): $10^{-3}$ meters. Used for larger organisms such as animals.

Example: Typical bacteria are measured in micrometers, while viruses are measured in nanometers.

Principles of Microscopy

• Total Magnification: The product of the objective lens magnification and the ocular (eyepiece) lens magnification. Formula: $\text{Total Magnification} = \text{Objective Magnification} \times \text{Ocular Magnification}$

• Resolution: The ability to distinguish two adjacent objects as separate entities. Resolution of light microscope: 0.2 micrometers ($0.2\ \mu m$).

• Contrast: The ability to distinguish the specimen from the surrounding background.

Types of Light Microscopy

• Light or Bright Field Microscopy:

  • Advantages: Easy to use, widely available, relatively inexpensive.

  • Disadvantages: Can recognize cells but not fine details; cells often lack contrast with water.

  • Application: Staining is commonly used to improve contrast.

• Phase Contrast Microscopy:

  • Uses a special condenser to increase contrast without staining.

  • Ideal for viewing live cells and internal structures.

• Dark Field Microscopy:

  • Specimens appear bright against a dark background.

  • Uses a special condenser with a disc to block direct light.

  • Useful for observing motility and morphology of organisms.

Example: Yeast (a fungus) under phase contrast microscopy

Yeast cells can be visualized without staining using phase contrast, revealing their internal structures and morphology.

Staining Techniques

Purpose of Staining

• Staining increases contrast, making cells and their components more visible under the microscope.

Types of Stains

• Simple Stain: Uses a single dye to color all cells uniformly.

• Differential Stain: Uses two or more dyes to distinguish between different types of cells or cell structures (e.g., Gram stain).

Gram Staining Procedure

1. Primary Stain: Crystal violet is applied to the smear.

2. Mordant: Iodine is added to help the stain adhere to cells.

3. Decolorization: Alcohol or acetone is used to remove the stain from Gram-negative cells.

4. Counterstain: Safranin is applied to stain Gram-negative cells.

Results:

• Gram-positive cells: Purple

• Gram-negative cells: Pink/red

Classification of Microorganisms

Taxonomy and Nomenclature

• Taxonomy: The science of classifying and naming organisms.

• Binomial Nomenclature: Developed by Linnaeus; each organism is given a genus and species name (e.g., Escherichia coli).

Hierarchical Classification

Level	Example
Domain	Bacteria, Archaea, Eukarya
Kingdom	Prokaryote
Phylum	Proteobacteria
Class	Gammaproteobacteria
Order	Enterobacteriales
Family	Enterobacteriaceae
Genus	Escherichia
Species	coli

Additional info:

• Classification helps in identifying, studying, and understanding relationships among microorganisms.

• Physical characteristics, biochemical tests, and nucleic acid analysis (e.g., DNA fingerprinting) are used for microbial identification.

Summary Table: Size Comparison of Biological Entities

Entity	Approximate Size	Unit
Atoms	0.1	nm
Proteins	5-10	nm
Viruses	20-300	nm
Bacteria	1-10	μm
Human red blood cell	7	μm
Flea	2	mm
Pig	1	m

Additional info: This table illustrates the relative sizes of biological entities, emphasizing the need for different types of microscopy to visualize them.