Microscopy, Staining, and Classification in Microbiology Chapter 4

Chapter 4: Microscopy, Staining, and Classification

Overview

This chapter introduces the fundamental concepts of microscopy, staining, and the classification of microorganisms, which are essential for the study and identification of microbes in microbiology.

Metric Units of Measurement

Metric System in Microbiology

The metric system is the standard system of measurement in science, including microbiology, due to its simplicity and suitability for measuring microscopic entities.

• Definition: The metric system is a decimal-based system of measurement used worldwide in scientific contexts.

• Common Units: Meter (m), millimeter (mm), micrometer (μm), nanometer (nm).

• Importance: Units are small enough to measure microscopic organisms accurately.

Prefix	Metric Equivalent	U.S. Equivalent
1 m	100 m	39.37 in
1/10	0.1 m = 10-1 m	3.94 in
1/100	0.01 m = 10-2 m	0.39 in = 2.54 cm
1/1,000	0.001 m = 10-3 m	1 mm
1/1,000,000	0.000001 m = 10-6 m	1 μm
1/1,000,000,000	0.000000001 m = 10-9 m	1 nm

Example: Bacteria are typically measured in micrometers (μm), while viruses are measured in nanometers (nm).

Microscopy

General Principles of Microscopy

Microscopy involves the use of light or electrons to magnify objects too small to be seen with the naked eye. Understanding the principles of microscopy is essential for observing microorganisms.

• Wavelength of Radiation: The distance between two corresponding points of a wave. Shorter wavelengths provide higher resolution.

• Magnification: The apparent increase in size of an object, indicated by a number followed by 'x' (times). Achieved by bending (refracting) light through lenses.

• Resolution (Resolving Power): The ability to distinguish between two points that are close together. Higher resolution allows for clearer images of small structures.

• Contrast: Differences in intensity between an object and its background. Enhanced by staining or using special optical techniques.

Key Equation:

$\text{Resolution} = \frac{0.61 \lambda}{\text{Numerical Aperture}}$

Where $\lambda$ is the wavelength of light used.

The Electromagnetic Spectrum and Microscopy

The electromagnetic spectrum includes all types of electromagnetic radiation, from gamma rays to radio waves. Visible light is used in light microscopy, while electron microscopes use electron beams with much shorter wavelengths for higher resolution.

• Visible Light: Wavelengths from approximately 400 nm (violet) to 700 nm (red).

• Shorter Wavelengths: (e.g., electrons) provide greater resolving power.

Magnification and Image Formation

Magnification occurs when light passes through a convex lens, bending and spreading the rays to produce an enlarged, inverted image of the specimen.

• Focal Point: The point where light rays converge after passing through a lens.

• Total Magnification: Product of the magnification of the objective lens and the ocular lens.

Example: If the objective lens is 40x and the ocular lens is 10x, total magnification is $40 \times 10 = 400x$.

Resolution and Clarity

Resolution is critical for distinguishing fine details. Magnifying an image beyond the resolving power of the microscope results in a blurry image with no additional useful information.

• Human Eye: Can resolve objects down to about 200 μm.

• Light Microscopes: Can resolve objects as small as 200 nm.

• Electron Microscopes: Can resolve objects as small as 0.1 nm.

Example: Viruses (20–300 nm) are visible only with electron microscopes, while bacteria (1–10 μm) can be seen with light microscopes.

Limits of Resolution: Human Eye vs. Microscopes

The ability to resolve small objects varies by instrument:

• Compound Light Microscope: 200 nm – 10 mm

• Scanning Electron Microscope (SEM): 4 nm – 1 mm

• Transmission Electron Microscope (TEM): 0.078 nm – 100 μm

• Scanning Tunneling Microscope (STM): 0.01 nm – 10 nm

• Atomic Force Microscope (AFM): 1 nm – 10 nm

Example: The diameter of a typical bacterium is about 1 μm, which is well within the resolving power of a light microscope.

Summary Table: Resolution Limits

Instrument	Resolution Limit	Objects Observable
Human Eye	~200 μm	Flea, chicken egg
Light Microscope	~200 nm	Bacteria, mitochondria, red blood cells
Electron Microscope	~0.1 nm	Viruses, proteins, DNA, atoms

Additional info: The resolving power of a microscope is determined by both the wavelength of the illuminating radiation and the numerical aperture of the lens system. Shorter wavelengths and higher numerical apertures yield better resolution.