Microscopy, Staining, and Classification in Microbiology

Microscopy, Staining, and Classification

Overview

This chapter introduces the fundamental concepts of microscopy, staining, and the classification of microorganisms. Understanding these topics is essential for observing, identifying, and categorizing microbes in the laboratory and clinical settings.

Microscopy

General Principles of Microscopy

Microscopy is the use of microscopes to view objects and areas of objects that cannot be seen with the naked eye. Several key principles determine the effectiveness of a microscope:

• Wavelength of Radiation: The distance between two consecutive peaks of a wave. Shorter wavelengths provide greater resolving power.

• Magnification: The process of enlarging the appearance of an object. It is the ratio of the image size to the actual size of the object.

• Resolution: The ability to distinguish two points that are close together. Higher resolution allows for clearer, more detailed images.

• Contrast: The difference in intensity between an object and its background. Contrast is crucial for distinguishing structures within a specimen.

Metric Units of Length

The metric system is used in microscopy to measure microorganisms. The standard unit is the meter (m), but smaller units are commonly used:

Unit	Prefix	Metric Equivalent	Representative Microbiological Application
Meter (m)	—	1 m	Length of some tapeworms
Millimeter (mm)	1/1,000	0.001 m = 10-3 m	Diameter of a bacterial colony
Micrometer (μm)	1/1,000,000	0.000001 m = 10-6 m	Size of most bacteria
Nanometer (nm)	1/1,000,000,000	0.000000001 m = 10-9 m	Size of viruses

Electromagnetic Spectrum and Microscopy

The electromagnetic spectrum encompasses all types of electromagnetic radiation, including visible light, ultraviolet (UV) light, and X-rays. Microscopes use different parts of this spectrum to visualize specimens:

• Visible light (400–700 nm) is used in light microscopy.

• Shorter wavelengths (e.g., electron beams) are used in electron microscopy for higher resolution.

Light Refraction and Image Magnification

Microscopes use lenses to bend (refract) light, focusing it to form magnified images of specimens. A convex lens converges light rays to a focal point, creating an enlarged, inverted image.

Resolution

• Definition: The minimum distance at which two points can be distinguished as separate entities.

• Formula: $d = \frac{0.61 \lambda}{n \sin \theta}$, where $d$ is the minimum resolvable distance, $\lambda$ is the wavelength, $n$ is the refractive index, and $\theta$ is the half-angle of the maximum cone of light that can enter the lens.

• Different microscopes have different resolving powers, with electron microscopes providing the highest resolution.

Limits of Resolution

The resolving power of the human eye and various microscopes determines what structures can be visualized:

• Human eye: ~200 μm

• Light microscope: ~200 nm

• Transmission electron microscope (TEM): ~0.1 nm

• Scanning electron microscope (SEM): ~10 nm

Example: Atoms and small molecules can only be seen with electron microscopes, while bacteria and eukaryotic cells can be seen with light microscopes.

Contrast

• Definition: The difference in light intensity between the specimen and the background.

• Contrast is essential for distinguishing structures within a specimen.

• Staining and the use of phase-contrast techniques can enhance contrast.

Types of Microscopes

Light Microscopy

• Bright-Field Microscopes: Use visible light to illuminate specimens. Can be simple (single lens) or compound (multiple lenses). Oil immersion increases resolution.

• Dark-Field Microscopes: Only scattered light enters the objective lens, making specimens appear bright against a dark background. Useful for observing pale or colorless cells.

• Phase Microscopes: Enhance contrast by exploiting differences in refractive index. Types include phase-contrast and differential interference contrast microscopes. Useful for live, unstained specimens.

• Fluorescence Microscopes: Use UV light to excite fluorescent dyes or naturally fluorescent specimens. Emitted light is detected, increasing resolution and contrast. Used in immunofluorescence to identify pathogens.

• Confocal Microscopes: Use lasers and fluorescent dyes to create sharp, 3D images by focusing on a single plane within the specimen.

Electron Microscopy

• Transmission Electron Microscope (TEM): Passes electrons through thin specimens, providing detailed images of internal structures. Resolution up to 0.1 nm.

• Scanning Electron Microscope (SEM): Scans the surface with electrons, producing 3D images of specimen surfaces. Resolution up to 10 nm.

Probe Microscopy

• Scanning Tunneling Microscope (STM): Measures electron flow between a probe and specimen surface, allowing visualization of atomic structures.

• Atomic Force Microscope (AFM): Uses a probe to scan the surface, providing topographical maps at atomic resolution.

Comparison of Microscopes

The following table summarizes the main features of different types of microscopes:

Microscope Type	Source of Illumination	Maximum Magnification	Resolution	Best Use
Bright-field (compound)	Visible light	~1,000x	~200 nm	Stained specimens, general observation
Dark-field	Visible light	~1,000x	~200 nm	Pale/colorless specimens
Phase-contrast	Visible light	~1,000x	~200 nm	Live, unstained specimens
Fluorescence	UV light	~1,000x	~200 nm	Fluorescently labeled cells
Confocal	Laser	~1,000x	~200 nm	3D imaging of thick specimens
TEM	Electron beam	100,000x+	~0.1 nm	Internal cell structures
SEM	Electron beam	100,000x+	~10 nm	Surface structures
STM/AFM	Probe	100,000,000x+	Atomic	Atomic-level imaging

Additional info: Table entries inferred and summarized for clarity and completeness.