Microscopy Fundamentals

Microscopy is essential for observing microorganisms that are smaller than the human eye's resolution of $0.2\,mm$ .
Common metric units used include: * 1 centimeter (cm): $10^{-2}\,m$ * 1 millimeter (mm): $10^{-3}\,m$ * 1 micrometer ( $\mu m$ ): $10^{-6}\,m$ * 1 nanometer (nm): $10^{-9}\,m$
Lenses refract (bend) light based on the refractive index of the media.
Lens magnification is determined by focal length; a shorter focal length results in higher magnification.

Magnification: Total magnification is calculated as: $\text{Ocular magnification} \times \text{Objective magnification}$ .
Resolution (Resolving Power): The capacity to distinguish two adjacent points. The light microscope limit is approximately $0.2\,\mu m$ .
Contrast: The difference in brightness or color between the specimen and the background.
Factors improving resolution include shorter light wavelengths and the use of immersion oil to increase numerical aperture.

Bright-Field Microscope: Dark image against a bright background; typically parfocal (stays in focus when switching objectives).
Dark-Field Microscope: Bright image against a dark background; ideal for living, unstained prep or identifying Treponema pallidum.
Phase-Contrast Microscope: Uses direct and diffracted light rays to observe internal structures in living organisms without staining.
Fluorescence Microscope: Uses UV or blue light to excite specimens stained with fluorochromes; emits longer visible wavelengths.
Confocal Microscopy: Employs laser scanning to create sharp two-dimensional planes that a computer reconstructs into 3-D images.

Electrons replace light as the illumination source, providing much higher resolution due to shorter wavelengths.
Transmission Electron Microscope (TEM): Electrons pass through thin, chemically fixed, metal-stained sections to show internal details.
Scanning Electron Microscope (SEM): Scans the surface of a specimen with excited electrons to create realistic 3-D images of surface features.

Fixation: Kills and attaches microbes to slides. Heat fixation is common for bacteria and archaea; chemical fixation is used for delicate organisms.
Basic Dyes ( $\text{positive charges}$ ) and Acid Dyes ( $\text{negative charges}$ ) bind to different cell components.
Differential Staining: * Gram Stain: Separates bacteria into Gram-positive and Gram-negative based on cell wall structure. * Acid-Fast Stain: Used for Mycobacterium tuberculosis and Mycobacterium leprae due to high mycolic acid content.
Structural Staining: Includes negative staining for capsules and the use of a mordant for flagella staining.