Microscopes

Principles of Microscopy

Microscopy is the use of light or electrons to magnify objects too small to be seen with the naked eye. It is fundamental in microbiology for observing cells, organelles, and viruses.

• Micrometer (µm): Unit used to measure the size of cells (1 µm = 10-6 meters).

• Nanometer (nm): Unit used to measure organelles and viruses (1 nm = 10-9 meters).

Electromagnetic Spectrum and Wavelength

• Wavelength: The distance between two corresponding parts of a wave. Determines the resolving power of a microscope.

• White light: Has an average wavelength of 550 nm.

Magnification and Image Formation

• Magnification: The apparent increase in the size of an object. Achieved by bending (refracting) light as it passes through lenses.

• Factors affecting magnification: Thickness and curvature of the lens, and the speed of light through the lens material.

• Image characteristics: Microscopes produce large, inverted images because of light refraction.

Resolution (Resolving Power)

• Resolution: The ability to distinguish two points that are close together. Also called resolving power.

• Historical context: Leeuwenhoek's microscope had a resolving power of approximately 1 µm; modern light microscopes achieve about 0.2 µm.

• Improvement: Use of shorter wavelength light and better lenses increases resolution.

Types of Light Microscopy

• Bright-field microscopy: The most common type; the field is illuminated and specimens appear darker.

• Dark-field microscopy: Specimens appear light against a dark background; useful for observing pale objects.

• Phase-contrast microscopy: Enhances contrast by aligning light waves; useful for observing living cells.

• Fluorescence microscopy: Uses ultraviolet (UV) light and fluorescent dyes to visualize specimens.

• Confocal microscopy: Uses lasers to illuminate fluorescent chemicals, producing sharp, three-dimensional images.

Oil Immersion Technique

• Oil immersion lens: Special lens that uses oil to capture more light, increasing resolution at high magnification.

Electron Microscopy

• Electron microscopes: Use beams of electrons instead of light, allowing magnification from 10,000X to 100,000X.

• Ultrastructure: Fine details of cells visible only with electron microscopes.

• Transmission Electron Microscope (TEM): Electrons pass through the specimen; magnetic fields act as lenses.

• Scanning Electron Microscope (SEM): Electrons scan the surface of the specimen, producing detailed surface images.

Staining Techniques

Preparation of Specimens

• Smear: A thin film of organisms spread on a microscope slide.

• Fixation: Process of attaching cells to the slide using heat (flame) or chemicals (e.g., methyl alcohol).

Dyes and Staining

• Dyes: Salts composed of a colored ion (chromophore) that bind to cellular components via covalent, ionic, or hydrogen bonds.

• Simple staining: Soak the smear in dye for 20–60 seconds, then rinse with water.

Gram Stain Procedure

• Gram-positive bacteria: Stain purple due to thick peptidoglycan layer.

• Gram-negative bacteria: Stain pink due to thinner cell wall and loss of primary stain during decolorization.

• Steps:

  1. Crystal violet (primary stain)

  2. Mordant (e.g., iodine) binds dye, making it less soluble

  3. Decolorizing agent (e.g., ethanol or 95% ethanol) removes stain from Gram-negative cells

  4. Counterstain (safranin) stains Gram-negative cells pink

• Result: Gram-positive cells appear purple; Gram-negative cells appear pink.

Negative Staining and Special Stains

• Negative stains: Acidic dyes repelled by negatively charged cell surfaces; stain the background, leaving cells colorless.

• Capsule staining: Encapsulated cells appear to have a halo.

• Flagella staining: Special stains are required to visualize thin flagella under light microscopy.

Classification of Microorganisms

Strains and Serotypes

• Strain: A population of cells that arose from a single cell; may have unique characteristics.

• Serotype: A strain distinguished by its unique set of antigens.

Biochemical and Structural Classification

• Ribosomes: Three basic types exist, differing in structure and function (e.g., prokaryotic 70S, eukaryotic 80S).

• Biochemical tests: Used to classify bacteria based on metabolic capabilities, such as:

  • Fermentation of various carbohydrates

  • Utilization of substrates (e.g., amino acids, starch, citrate, gelatin)

  • Production of waste products (e.g., hydrogen sulfide gas, H2S)

  • Differences in fatty acid composition

Summary Table: Types of Microscopy

Key Equations

• Resolution (d):

• Where d is the minimum resolvable distance, λ is the wavelength of light, n is the refractive index, and θ is the half-angle of the maximum cone of light that can enter the lens.

Additional info: The above equation is the Abbe equation for resolution, which explains why shorter wavelengths and higher numerical apertures improve resolving power.